ABSTRACT
Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.
Subject(s)
Creatinine/analogs & derivatives , Toxicity Tests, Chronic , Administration, Oral , Animals , Creatinine/administration & dosage , Creatinine/pharmacokinetics , Creatinine/toxicity , Dogs , Dose-Response Relationship, Drug , Female , Half-Life , Kidney/drug effects , Kidney/pathology , Lung/drug effects , Lung/pathology , Lung/physiopathology , Lung Diseases/chemically induced , Lung Diseases/pathology , Lung Diseases/physiopathology , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Muscle, Smooth/drug effects , Muscle, Smooth/pathology , Myocardium/pathology , No-Observed-Adverse-Effect Level , Risk Assessment , Time Factors , Toxicokinetics , Vacuoles/drug effects , Vacuoles/pathologyABSTRACT
In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.
Subject(s)
Antineoplastic Agents/toxicity , Creatinine/analogs & derivatives , Disease Models, Animal , Animals , Brain , Brain Diseases, Metabolic, Inborn , Creatine/deficiency , Creatinine/toxicity , Humans , Membrane Transport Proteins , Mental Retardation, X-Linked , Mice , No-Observed-Adverse-Effect Level , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Seizures , Tissue DistributionABSTRACT
New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.
Subject(s)
Blood Platelets/metabolism , Hematopoiesis , Megakaryocytes/metabolism , Polyploidy , Thrombocytopenia/metabolism , Tyrosine-tRNA Ligase/metabolism , Blood Platelets/pathology , Cell Culture Techniques , Cells, Cultured , Female , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Male , Megakaryocytes/pathology , Signal Transduction , Thrombocytopenia/pathology , Thrombopoietin/metabolismABSTRACT
Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.
Subject(s)
Brain/drug effects , Creatinine/analogs & derivatives , Toxicity Tests, Chronic/methods , Administration, Oral , Animals , Brain/metabolism , Brain/pathology , Creatine/analogs & derivatives , Creatine/blood , Creatine/toxicity , Creatinine/administration & dosage , Creatinine/blood , Creatinine/toxicity , Female , Male , Organ Size/drug effects , Organ Size/physiology , Rats , Rats, Sprague-Dawley , Testis/drug effects , Testis/metabolism , Testis/pathology , Thyroid Gland/drug effects , Thyroid Gland/metabolism , Thyroid Gland/pathology , Time FactorsABSTRACT
CONTEXT: Recombinant human growth hormone (rhGH) is approved for treatment of pediatric growth hormone deficiency (GHD), with greatest growth responses observed in those with severe GHD. Orally administered GH secretagogues (GHS) may be useful treatment in patients with moderate GHD. Distinguishing children with severe vs moderate GHD could identify children who would be better treated with rhGH or GHS. OBJECTIVES: Evaluate baseline insulin-like growth factor-I (IGF-I) and stimulated peak GH response as predictors of 12-month height velocity (HV) in children with GHD. DESIGN: Data on children with GHD were analyzed in a legacy data base (GeNeSIS data). PARTICIPANTS: 514 naïve to rhGH-treatment, prepubertal children with idiopathic isolated GHD for whom stimulated GH, baseline serum IGF-I, and first-year HV during rhGH treatment data are available. OUTCOME MEASURES: Children with severe or moderate GHD were categorized based on GH and IGF-I data and evaluated based on baseline auxologic and hormone profiles and first-year growth response to rhGH. RESULTS: Cohorts of severe and moderate GHD were 81/514 (15.8%) and 433/514 (84.2%). Cohorts differed significantly with regard to indicators of GHD [eg, baseline height SD score (SDS), height SDS minus target height SDS, HV, HV SDS, and change in height SDS during rhGH treatment]. Multiple regression analysis showed IGF-I and stimulated GH were significant predictors of HV independent of other known variables. Expected first-year HV in moderate GHD was 8.3 cm/y. CONCLUSIONS: The combination of peak GH to GH stimulation testing and baseline IGF-I concentration are predictive enrichment markers for annualized HV responses to rhGH therapy.
ABSTRACT
CONTEXT: We hypothesize, based on the degree of residual hypothalamic-pituitary function, that some, but not all, children with growth hormone deficiency (GHD) may have beneficial growth responses to the orally administered growth hormone (GH) secretagogue LUM-201. OBJECTIVE: To determine if pretreatment testing can identify predictive enrichment markers (PEM) for subjects with adequate residual function who are responsive to LUM-201. METHODS: We performed an analysis of a completed, randomized, placebo-controlled trial of LUM-201, a GH secretagogue receptor agonist, in which all randomized subjects had pretreatment testing. This international multicenter study conducted in pediatric endocrinology clinics included 68 naïve-to-treatment, prepubertal children with established diagnoses of GHD. Outcome measures included the sensitivity, specificity, and predictive accuracy of potential markers to predict 6-month growth responses to oral LUM-201 and daily rhGH. RESULTS: Two PEM were identified for use in defining PEM-positive status: (1) baseline insulin-like growth factor I (IGF-I) concentration >30 ng/mL and (2) peak GH response of ≥5 ng/mL upon administration of single-dose LUM-201. PEM-positive status enriches a population for better growth responses to LUM-201. PEM-negative status enriches a population for better growth responses to rhGH. CONCLUSION: Combined, the peak GH response to single-dose LUM-201 and the baseline IGF-I concentration are effective PEMs for 6-month growth responses to LUM-201 and rhGH in prepubertal children with GHD.
ABSTRACT
The neuropeptide GNRH 1 stimulates the secretion of the reproductive hormone LH in pituitary gonadotropes. Other secretory cell types depend on the unfolded protein response (UPR) pathway to regulate protein synthesis and protect against endoplasmic reticulum (ER) stress in response to differentiation or secretory stimuli. This study investigated the role of the UPR in GNRH action within the LbetaT2 gonadotrope model. Cells were treated with GNRH, and the activation of UPR signaling components and general translational status was examined. The ER-resident stress sensors, Atf6, Eif2ak3, and Ern1, are all present, and GNRH stimulation results in the phosphorylation of eukaryotic translation initiation factor 2A kinase 3 and its downstream effector, eukaryotic translation initiation factor 2A. Additionally, activation of the UPR was confirmed both in LbetaT2 as well as mouse primary pituitary cells through identifying GNRH-induced splicing of Xbp1 mRNA, a transcription factor activated by splicing by the ER stress sensor, ER to nucleus signaling 1. Ribosome profiling revealed that GNRH stimulation caused a transient attenuation in translation, a hallmark of the UPR, remodeling ribosomes from actively translating polysomes to translationally inefficient ribonucleoprotein complexes and monosomes. The transient attenuation of specific mRNAs was also observed. Overall, the results show that GNRH activates components of the UPR pathway, and this pathway may play an important physiological role in adapting the ER of gonadotropes to the burden of their secretory demand.
Subject(s)
Gonadotrophs/drug effects , Gonadotrophs/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Animals , Base Sequence , Calcium/metabolism , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Gonadotrophs/cytology , Mice , Models, Biological , Protein Folding/drug effects , RNA Splicing/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulatory Factor X Transcription Factors , Ribonucleoproteins/metabolism , Signal Transduction/drug effects , Stress, Physiological/drug effects , Transcription Factors/genetics , Transcription Factors/metabolism , X-Box Binding Protein 1 , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolismABSTRACT
Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr.
Subject(s)
Autistic Disorder/etiology , Brain Diseases, Metabolic, Inborn/drug therapy , Cognition Disorders/etiology , Creatine/deficiency , Creatinine/analogs & derivatives , Epilepsy/etiology , Mental Retardation, X-Linked/drug therapy , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Animals , Autistic Disorder/drug therapy , Blood-Brain Barrier , Brain Diseases, Metabolic, Inborn/complications , Cerebrovascular Circulation/drug effects , Cognition Disorders/drug therapy , Creatinine/therapeutic use , Disease Models, Animal , Electroencephalography , Epilepsy/drug therapy , Hemodynamics/drug effects , Male , Mental Retardation, X-Linked/complications , Mice , Mice, Inbred C57BL , Phenotype , Seizures/drug therapy , Seizures/etiology , Stereotyped Behavior/drug effectsABSTRACT
Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.
Subject(s)
Brain Diseases, Metabolic, Inborn/drug therapy , Creatine/deficiency , Creatinine/analogs & derivatives , Fibroblasts/metabolism , Imidazolidines/metabolism , Mental Retardation, X-Linked/drug therapy , Phosphocreatine/analogs & derivatives , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Brain Diseases, Metabolic, Inborn/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Creatine/metabolism , Creatinine/pharmacokinetics , Creatinine/therapeutic use , Humans , Imidazolidines/analysis , Mental Retardation, X-Linked/metabolism , Phosphocreatine/analysis , Phosphocreatine/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Primary Cell Culture , Tandem Mass Spectrometry/methodsABSTRACT
The study of gene expression in gonadotropes has largely focused on the variety of mechanisms regulating transcription of the gonadotropin genes and ancillary factors that contribute to the overall phenotype and function of these cells in reproduction. However, there are aspects of the response to GNRH signaling that are not readily explained by changes at the level of transcription. As our understanding of regulation at the level of mRNA translation has increased, it has become evident that GNRH receptor signaling engages multiple aspects of translational regulation. This includes activation of cap-dependent translation initiation, translational pausing caused by the unfolded protein response and RNA binding protein interaction. Gonadotropin mRNAs and the mRNAs of other factors that control the transcriptional and signaling responses to GNRH have been identified as targets of regulation at the level of translation. In this review we examine the impact of translational control of the expression of gonadotropin genes and other genes relevant to GNRH-mediated control of gonadotrope function.
Subject(s)
Gene Expression Regulation/physiology , Gonadotrophs/metabolism , Peptide Chain Initiation, Translational/physiology , Signal Transduction/physiology , Unfolded Protein Response/physiology , Animals , Gonadotrophs/cytology , Humans , Receptors, LHRH/metabolismABSTRACT
The neuropeptide gonadotropin-releasing hormone stimulates synthesis and secretion of the glycoprotein gonadotropic hormones and activates the unfolded protein response, which causes a transient reduction of endoplasmic reticulum-associated mRNA translation. Hormone-treated cell extracts were fractionated to resolve mRNA in active polyribosomes from mRNA in inactive complexes. Quantitative real-time PCR and expression array analysis were used to determine hormone-induced redistribution of mRNAs between fractions and individual mRNAs were found to be redistributed differentially. Among the affected mRNAs relevant to gonadotropin synthesis, the luteinizing hormone subunit genes Lhb and Cga were enriched in the ribonucleoprotein pool. The MAP kinase phosphatase Dusp1 was enriched in the polyribosome pool. Enrichment of Dusp1 mRNA in the polyribosome pool was independent of the unfolded protein response, sensitive to ERK inhibition, and dependent on the 3'untranslated region. The results show that GnRH exerts translational control to modulate physiologically relevant gene expression through two distinct signaling pathways.
Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , MAP Kinase Signaling System/drug effects , Polyribosomes/metabolism , Ribonucleoproteins/metabolism , eIF-2 Kinase/metabolism , 3' Untranslated Regions/genetics , Animals , Cell Line , Dual Specificity Phosphatase 1/genetics , Dual Specificity Phosphatase 1/metabolism , ELAV Proteins/metabolism , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Mice , Protein Biosynthesis/drug effects , Protein Kinase C/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sirolimus/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Unfolded Protein Response/drug effectsABSTRACT
Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Antigens, CD/metabolism , Cadherins/metabolism , Catalytic Domain , Models, Molecular , Tryptophan/metabolism , Amino Acyl-tRNA Synthetases/genetics , Blotting, Western , Humans , Immunoprecipitation , Mutagenesis , Structure-Activity RelationshipABSTRACT
The integrated signaling of insulin and gonadotropin-releasing hormone in the pituitary gonadotropes may have a profound bearing on reproductive function, although the cross-receptor signaling mechanisms are unclear. We demonstrate that the insulin receptor is constitutively localized to non-caveolar lipid raft microdomains in the pituitary gonadotrope cell line LbetaT2. The localization to rafts is consistent with similar localization of the GnRH receptor. Insulin receptor phosphorylation occurs in raft domains and activates the downstream signaling targets Insulin Receptor Substrate1 and Akt/Protein Kinase B. Although insulin alone does not strongly activate the extracellular signal-regulated kinase second messenger cascade, co-stimulation potentiates the phosphorylation of the extracellular signal-regulated kinase by gonadotropin-releasing hormone. The co-stimulatory effect of insulin and gonadotropin-releasing hormone is also evident in increased activation of cap-dependent translation. In contrast, co-stimulation attenuates Akt/Protein Kinase B activation. Our results show that both gonadotropin-releasing hormone and insulin are capable of mutually altering their respective regulatory signaling cascades. We suggest that this provides a mechanism to integrate neuropeptide and energy homeostatic signals to modulate reproductive function.