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1.
J Pharmacol Exp Ther ; 377(1): 11-19, 2021 04.
Article in English | MEDLINE | ID: mdl-33509901

ABSTRACT

Gain-of-function mutations in leucine-rich kinase 2 (LRRK2) are associated with increased incidence of Parkinson disease (PD); thus, pharmacological inhibition of LRRK2 kinase activity is postulated as a disease-modifying treatment of PD. Histomorphological changes in lungs of nonhuman primates (NHPs) treated with small-molecule LRRK2 kinase inhibitors have brought the safety of this treatment approach into question. Although it remains unclear how LRRK2 kinase inhibition affects the lung, continued studies in NHPs prove to be both cost- and resource-prohibitive. To develop a tractable alternative animal model platform, we dosed male mice in-diet with the potent, highly selective LRRK2 kinase inhibitor MLi-2 and induced histomorphological changes in lung within 1 week. Oral bolus dosing of MLi-2 at a frequency modeled to provide steady-state exposure equivalent to that achieved with in-diet dosing induced type II pneumocyte vacuolation, suggesting pulmonary changes require sustained LRRK2 kinase inhibition. Treating mice with MLi-2 in-diet for up to 6 months resulted in type II pneumocyte vacuolation that progressed only modestly over time and was fully reversible after withdrawal of MLi-2. Immunohistochemical analysis of lung revealed a significant increase in prosurfactant protein C staining within type II pneumocytes. In the present study, we demonstrated the kinetics for onset, progression, and rapid reversibility of chronic LRRK2 kinase inhibitor effects on lung histomorphology in rodents and provide further evidence for the derisking of safety and tolerability concerns for chronic LRRK2 kinase inhibition in PD. SIGNIFICANCE STATEMENT: We have defined a mouse model by which the on-target lung effects of leucine-rich kinase 2 (LRRK2) kinase inhibition can be monitored, whereas previous in vivo testing relied solely on nonhuman primates. Data serve to derisk long-term treatment with LRRK2 kinase inhibitors, as all lung changes were mild and readily reversible.


Subject(s)
Alveolar Epithelial Cells/drug effects , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , Animals , Indazoles/administration & dosage , Indazoles/pharmacology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Male , Mice , Mice, Inbred C57BL , Morpholines/administration & dosage , Morpholines/pharmacology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Pulmonary Surfactant-Associated Protein C/genetics , Pulmonary Surfactant-Associated Protein C/metabolism , Pyrimidines/administration & dosage , Pyrimidines/pharmacology
2.
Mol Cancer Ther ; 19(6): 1298-1307, 2020 06.
Article in English | MEDLINE | ID: mdl-32229606

ABSTRACT

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/pharmacokinetics , Leukocytes, Mononuclear/drug effects , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocytes/drug effects , Animals , Antineoplastic Agents, Immunological/pharmacokinetics , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Female , Humans , Immune Checkpoint Inhibitors/pharmacokinetics , Immune Checkpoint Inhibitors/pharmacology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Macaca fascicularis , Mice , Mice, Inbred BALB C , Neoplasms/immunology , Neoplasms/pathology , Programmed Cell Death 1 Ligand 2 Protein/antagonists & inhibitors , Programmed Cell Death 1 Ligand 2 Protein/immunology , Programmed Cell Death 1 Ligand 2 Protein/metabolism , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tissue Distribution , Toxicity Tests
3.
Birth Defects Res B Dev Reprod Toxicol ; 107(2): 108-19, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27062127

ABSTRACT

This report discusses the principles of reproductive toxicity risk assessment for biopharmaceuticals blocking the PD-1/programmed cell death ligand 1 (PD-L1) pathway, which have been developed for the treatment of patients with advanced malignancies. The PD-1/PD-L1 pathway is a T-cell co-inhibitory pathway that normally maintains immune tolerance to self. Its role in pregnancy is to maintain immune tolerance to the fetal allograft. In cancer patients, this signaling pathway is hijacked by some neoplasms to avoid immune destruction. PD-1/PD-L1-blocking agents enhance functional activity of the target lymphocytes to eventually cause immune rejection of the tumor. A therapeutic blockade of PD-1/PD-L1 pathway that occurs at full target engagement provides a unique challenge to address the risk to pregnancy because disruption of the same pathway may also reduce or abrogate maternal immune tolerance to the fetal alloantigens inherited through the father. Typically, nonclinical reproductive and developmental toxicity (DART) studies in animals (rats and rabbits) with clinical drug candidates are conducted to identify potential risk in humans and to determine exposure margin for the effects on reproduction as part of the risk assessment. However, for biopharmaceuticals for which the desired mechanism of action cannot be separated from potential deleterious effects to the fetus and when the only relevant toxicology species is nonhuman primate (NHP), the risk to reproduction can be predicted by a mechanism-based assessment using data generated from murine surrogate models as supportive information without conducting DART in NHPs. Such an approach has been used in the evaluation of pregnancy risk of anti-PD-1 agent, pembrolizumab, and has been demonstrated as an important alternative to performing DART studies in NHPs.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , B7-H1 Antigen/metabolism , Reproduction/drug effects , Animals , B7-H1 Antigen/antagonists & inhibitors , Disease Models, Animal , Drug Evaluation, Preclinical , Drug-Related Side Effects and Adverse Reactions/diagnosis , Female , Humans , Immunotherapy , Mice , Neoplasms/drug therapy , Placenta/drug effects , Placenta/metabolism , Pregnancy , Risk Assessment , Signal Transduction , Xenograft Model Antitumor Assays
4.
J Pharmacol Exp Ther ; 355(3): 397-409, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26407721

ABSTRACT

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of familial and sporadic Parkinson's disease (PD). That the most prevalent mutation, G2019S, leads to increased kinase activity has led to a concerted effort to identify LRRK2 kinase inhibitors as a potential disease-modifying therapy for PD. An internal medicinal chemistry effort identified several potent and highly selective compounds with favorable drug-like properties. Here, we characterize the pharmacological properties of cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2), a structurally novel, highly potent, and selective LRRK2 kinase inhibitor with central nervous system activity. MLi-2 exhibits exceptional potency in a purified LRRK2 kinase assay in vitro (IC50 = 0.76 nM), a cellular assay monitoring dephosphorylation of LRRK2 pSer935 LRRK2 (IC50 = 1.4 nM), and a radioligand competition binding assay (IC50 = 3.4 nM). MLi-2 has greater than 295-fold selectivity for over 300 kinases in addition to a diverse panel of receptors and ion channels. Acute oral and subchronic dosing in MLi-2 mice resulted in dose-dependent central and peripheral target inhibition over a 24-hour period as measured by dephosphorylation of pSer935 LRRK2. Treatment of MitoPark mice with MLi-2 was well tolerated over a 15-week period at brain and plasma exposures >100× the in vivo plasma IC50 for LRRK2 kinase inhibition as measured by pSer935 dephosphorylation. Morphologic changes in the lung, consistent with enlarged type II pneumocytes, were observed in MLi-2-treated MitoPark mice. These data demonstrate the suitability of MLi-2 as a compound to explore LRRK2 biology in cellular and animal models.


Subject(s)
Antiparkinson Agents/adverse effects , Antiparkinson Agents/therapeutic use , Indazoles/pharmacology , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/pathology , Animals , Behavior, Animal/drug effects , Binding, Competitive , Brain/metabolism , Brain Chemistry/drug effects , Cell Line , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Dose-Response Relationship, Drug , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Mutation/genetics , Parkinson Disease/drug therapy , Parkinson Disease/pathology , Parkinson Disease/psychology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism
5.
Toxicol Pathol ; 38(6): 907-22, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20805317

ABSTRACT

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.


Subject(s)
Acanthocytes/metabolism , Anemia, Hemolytic/chemically induced , Hematopoiesis, Extramedullary/drug effects , Membrane Lipids/metabolism , Receptors, CXCR3/antagonists & inhibitors , Thrombocytopenia/chemically induced , Acanthocytes/pathology , Anemia, Hemolytic/metabolism , Anemia, Hemolytic/pathology , Animals , Blood Platelets/drug effects , Blood Platelets/pathology , Cholesterol/metabolism , Erythrocyte Membrane/metabolism , Hypercholesterolemia/blood , Hypercholesterolemia/chemically induced , Osmotic Fragility , Phosphatidylcholines/metabolism , Rats , Sphingomyelins/metabolism , Spleen/drug effects , Spleen/metabolism , Spleen/pathology , Thrombocytopenia/metabolism , Thrombocytopenia/pathology , Triglycerides/blood
6.
Toxicol Pathol ; 38(4): 568-82, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20460440

ABSTRACT

This report describes the findings of preclinical testing of SCH 351591, a selective phosphodiesterase 4 inhibitor, in CD-1 mice over a wide range of doses, in which the heart and reproductive organs of both sexes demonstrated toxic effects. Repeat-dose toxicity studies assessed 5, 15, 50, 100, 200, 400, and 800 mg/kg/day, orally by gavage, for one or three months. Findings included higher testes and ovary weights and lower uterus weights (> or =200 mg/kg), small ovaries/uterus (> or =400 mg/kg), and histopathologic changes of large corpora lutea and ovarian atrophy at 200 and 800 mg/kg, respectively. In addition, chronic myocardial inflammation of the heart base occurred at 100 mg/kg. Vaginal staging of the estrous cycle revealed persistent diestrus. There was no histopathologic correlate or morphometric change to explain higher testes weights. A pilot fertility and early embryonic developmental toxicity study assessing doses of 100, 200, 400, and 800 mg/kg/day produced complementary results. Females had prolonged or abnormal estrous cycles, fewer successful pregnancies, increased ovarian corpora lutea, and decreased size of live litters owing to fetal resorptions. Male fertility was not affected. However, males had a 25% increase in testes weights at all doses. The pharmacology of specific PDE4 isoenzymes may explain both the reproductive and cardiac findings.


Subject(s)
Cyclic N-Oxides/toxicity , Heart Diseases/chemically induced , Infertility/chemically induced , Phosphodiesterase 4 Inhibitors , Quinolines/toxicity , Reproduction/drug effects , Animals , Diestrus/drug effects , Embryo, Mammalian , Female , Heart/drug effects , Histocytochemistry , Male , Mice , Myocardium/pathology , Organ Size/drug effects , Ovary/anatomy & histology , Ovary/drug effects , Testis/anatomy & histology , Testis/drug effects , Toxicity Tests, Chronic/methods , Uterus/anatomy & histology , Uterus/drug effects
7.
Toxicol Pathol ; 33(3): 320-8, 2005.
Article in English | MEDLINE | ID: mdl-15814361

ABSTRACT

Spontaneous hypospadias is seldom observed in rats in contrast to its occurrence in 1 out of 250 human births. Ziracin, an antibacterial of the everninomycin class under development for serious enterococcal, staphylococcal, and streptococcal infections, caused anomalies of the external genitalia in F1 female rats and decreased reproductive performance. To characterize the urogenital malformations and determine the period of sensitivity to the effects of Ziracin during development, pregnant rats (F0) were administered 60 mg/kg IV of Ziracin from GD6 to LD21, GD6 to 13, GD14 to the last day of gestation or LD0 to 21. Controls received saline or placebo from GD6 to LD21. Ziracin-induced changes occurred in F1 rats exposed from GD6 to LD21 and GD14 to the last day of gestation, indicating that the period of sensitivity to Ziracin was from GD 14 to the last day of gestation. The urogenital abnormalities consisted of cranial displacement of the urethral opening within the vagina from its normal location at the tip of the genital tubercle. When the urethrovaginal junction occurred at the distal third of the vagina, it created an urogenital cloaca. As a result, ascending infections were seen in the urinary and genital tract. No differences in survivability, body weight, and date of vaginal opening were observed in F1 females. The estrous cycles were slightly prolonged. The mating and fertility indices were decreased as a result of the urogenital anomalies. The mammary glands of pregnant F1 females were underdeveloped, thus F2 pups from affected F1 females had a decreased survival rate. Although the cause of these effects is not known, the findings are consistent with a potential hormonal mechanism.


Subject(s)
Aminoglycosides/toxicity , Genitalia, Female/pathology , Prenatal Exposure Delayed Effects , Teratogens/toxicity , Urogenital Abnormalities/pathology , Animals , Estrus/drug effects , Female , Fertility/drug effects , Genitalia, Female/drug effects , Pregnancy , Rats , Rats, Sprague-Dawley , Reproduction/drug effects , Urogenital Abnormalities/chemically induced
8.
Toxicol Pathol ; 32(5): 558-66, 2004.
Article in English | MEDLINE | ID: mdl-15603540

ABSTRACT

Phentolamine is a reversible competitive alpha-adrenergic antagonist with similar affinities for alphal and alpha2 receptors. It has a long history of safe clinical use, and was developed as a potential therapy for male erectile dysfunction because of its capacity to increase the arteriolar blood flow to the corpora cavernosa. Phentolamine mesylate was administered to rats by oral gavage at daily doses of 10, 50, and 150 mg/kg for 24 months. A dose-related increase in mortality, ascribed to an exaggerated pharmacologic effect, was seen at high doses. Systemic exposure as measured by plasma drug concentration increased with dose and duration of dosing and slight drug accumulation occurred, particularly in high-dose males. In the treated groups, 10 males and 1 female were diagnosed with hibernomas, neoplasms of brown adipose tissue, which appeared in the thoracic cavity or retroperitoneal area as circumscribed, tan to reddish-brown lobulated masses. Histologically, the masses were well circumscribed with variably sized lobules defined by a rich capillary network and consisted of closely apposed oval to polygonal cells with large amounts of cytoplasm and a centrally located nucleus. The cytoplasm's appearance varied from multivacuolated to univacuolated to granular eosinophilic. In a few cases, neoplastic emboli were observed in capsular vessels. Ultrastructurally, the neoplastic cells contained numerous mitochondria with transverse parallel cristae that occupied over 60% of the cytoplasm and lipid droplets. This study documents the previously unreported development of hibernomas in rats treated with phentolamine mesylate.


Subject(s)
Adrenergic alpha-Antagonists/toxicity , Carcinogens/toxicity , Lipoma/chemically induced , Phentolamine/toxicity , Retroperitoneal Neoplasms/chemically induced , Thoracic Neoplasms/chemically induced , Administration, Oral , Adrenergic alpha-Antagonists/administration & dosage , Adrenergic alpha-Antagonists/blood , Animals , Carcinogenicity Tests , Carcinogens/administration & dosage , Dose-Response Relationship, Drug , Female , Lipoma/pathology , Male , Mitochondria/drug effects , Mitochondria/ultrastructure , Phentolamine/administration & dosage , Phentolamine/blood , Rats , Rats, Sprague-Dawley , Retroperitoneal Neoplasms/pathology , Thoracic Neoplasms/pathology
9.
J Biol Chem ; 279(13): 12876-82, 2004 Mar 26.
Article in English | MEDLINE | ID: mdl-14709552

ABSTRACT

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic beta-amyloid (Abeta) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other gamma-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by gamma-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of gamma-secretase inhibition, the gamma-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Abeta production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4(-)CD8(-)CD44(+)CD25(+) precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak gamma-secretase inhibitor. These studies show that inhibition of gamma-secretase has the expected benefit of reducing Abeta in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing.


Subject(s)
Amyloid beta-Peptides/chemistry , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Peptides/chemistry , Administration, Oral , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Brain/drug effects , Cell Differentiation , Cell Division/drug effects , Cell Line , Cell Separation , Flow Cytometry , Humans , Lymphocytes/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Models, Chemical , Protein Binding , Receptors, Notch , T-Lymphocytes/cytology , Thymus Gland/pathology , Time Factors
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