Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 µM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Cynomolgus macaque model of neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degeneration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a homozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

Network pharmacology-based prediction of inhibiting leukocyte recruitment and angiogenesis of total glucosides of peony against rheumatoid arthritis.

BACKGROUND: Total glucosides of peony (TGP) is extracted from Paeonia lactiflora Pallas, which has been approved for rheumatoid arthritis (RA) treatment. There were approximately 15 monoterpene glycosides identified in TGP. Pervious researches focused on the effects of TGP and the major ingredient paeoniflorin (PF), but the functions of other monoterpene glycosides and their interactions were not clear. Network pharmacology has been one of the new strategies for multi-target drug discovery. In this study, we investigate the functions of all components of TGP and their interactions in RA treatment based on network pharmacology methods. METHODS: The components of TGP were searched out the Web of Science, PubMed, China National Knowledge Infrastructure databases; then we identified the potential targets based of chemical similarity in the Similarity Ensemble Approach. The molecular related with RA were obtained from DrugBank, GeneCards, DisGeNET and Online Mendelian Inheritance in Man (OMIM) databases. The components-targets-disease network was constructed and analyzed with Cytoscape software; Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted with R for function analysis. The hub components-targets interactions were validated with Autodock Vina. RESULTS: Twenty potential targets of TGP were predicted for RA treatment. The major components of TGP, PF and albiflorin (AF) had more predicted targets. Hub targets of TGP were LGALS3/9, VEGFA, FGF1, FGF2, IL-6, IL-2, SELP, PRKCA and ERAP1. These targets ameliorated RA mainly through inhibiting leukocyte recruitment and angiogenesis. Enriched pathways including VEGFR pathway, signaling by interleukins, PI3K-Akt signaling pathway, platelet activation, extracellular matrix organization, and so on. The combination of PF, AF and lactiflorin (LF) with the hub targets was further validated using docking program. CONCLUSIONS: We investigated the comprehensive mechanism of TGP for RA treatment. We analyzed the different targets of the components in TGP and predicted the new effects of TGP on inhibiting leukocyte recruitment and angiogenesis. This study provides a better understanding of TGP on the RA treatment.

ERAP2 as a potential biomarker for predicting gemcitabine response in patients with pancreatic cancer.

OBJECTIVE: Pancreatic cancer is one of the most malignant tumors, with rapid metastasis, high mortality rate, and difficult early screening. Currently, gemcitabine is a first-line drug for pancreatic cancer patients, but its clinical effect is limited due to drug resistance. It is particularly important to further identify biomarkers associated with gemcitabine resistance to improve the sensitivity of gemcitabine treatment. METHODS: Drug sensitivity data and the corresponding transcript data derived from the Genomics of Drug Sensitivity in Cancer (GDSC) database for correlation analysis was adopted to obtain genes related to gemcitabine sensitivity. Moreover, the survival model of pancreatic cancer patients treated with gemcitabine in The Cancer Genome Atlas (TCGA) database was utilized to obtain key genes. Multiple in vitro assays were performed to verify the function of the key biomarker. RESULTS: Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) was identified as a biomarker promoting gemcitabine resistance, and its high expression resulted in a worse prognosis. Besides, gemcitabine significantly increased the mRNA and protein levels of ERAP2 in pancreatic cancer cells. Additionally, ERAP2 knockdown suppressed tumorigenesis and potentiated gemcitabine-induced growth, migration and invasion inhibition in human pancreatic cancer cells. CONCLUSIONS: ERAP2 may be a novel key biomarker for gemcitabine sensitivity and diagnosis, thus providing an effective therapeutic strategy for pancreatic cancer treatment.

A Novel Porcine Model of CLN2 Batten Disease that Recapitulates Patient Phenotypes.

CLN2 Batten disease is a lysosomal disorder in which pathogenic variants in CLN2 lead to reduced activity in the enzyme tripeptidyl peptidase 1. The disease typically manifests around 2 to 4 years of age with developmental delay, ataxia, seizures, inability to speak and walk, and fatality between 6 and 12 years of age. Multiple Cln2 mouse models exist to better understand the etiology of the disease; however, these models are unable to adequately recapitulate the disease due to differences in anatomy and physiology, limiting their utility for therapeutic testing. Here, we describe a new CLN2R208X/R208X porcine model of CLN2 disease. We present comprehensive characterization showing behavioral, pathological, and visual phenotypes that recapitulate those seen in CLN2 patients. CLN2R208X/R208X miniswine present with gait abnormalities at 6 months of age, ERG waveform declines at 6-9 months, vision loss at 11 months, cognitive declines at 12 months, seizures by 15 months, and early death at 18 months due to failure to thrive. CLN2R208X/R208X miniswine also showed classic storage material accumulation and glial activation in the brain at 6 months, and cortical atrophy at 12 months. Thus, the CLN2R208X/R208X miniswine model is a valuable resource for biomarker discovery and therapeutic development in CLN2 disease.

Alpiniae oxyphyllae fructus polysaccharide 3 inhibits porcine epidemic diarrhea virus entry into IPEC-J2 cells.

Porcine epidemic diarrhea virus (PEDV) is deadly for suckling piglets and is a significant threat to most pig farms. Alpiniae oxyphyllae fructus polysaccharide 3 (AOFP3) shows antiviral activity against PEDV. However, the anti-PEDV mechanism of AOFP3 is unknown. Entering the host cell is important for viral infection, and many drugs play antiviral roles by inhibiting this process. To understand the antiviral mechanism of AOFP3 against PEDV, the effect of AOFP3 on PEDV entering IPEC-J2 cells was investigated in the present study. Real-time PCR and immunofluorescence were used to study the effect of AOFP3 on PEDV binding and penetrating IPEC-J2 cells. The effect of PEDV on AOFP3 attachment to IPEC-J2 cells was also investigated. Afterward, the effect of AOFP3 on PEDV spike (S) protein binding to porcine aminopeptidase was tested by using coimmunoprecipitation, and the effect of AOFP3 on the cholesterol level of IPEC-J2 cells was detected. The results showed that AOFP3 competitively inhibited PEDV adsorption on IPEC-J2 cells by blocking PEDV S protein binding to porcine aminopeptidase in IPEC-J2 cells. Furthermore, AOFP3 decreased PEDV penetration into host cells by decreasing the cholesterol level in IPEC-J2 cells.

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells.

RATIONALE: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells. METHODS: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays. RESULTS: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growth Conclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

Immunopathogenesis of Behçet's disease and treatment modalities.

INTRODUCTION: Behçet's disease (BD) is an auto-inflammatory disease, primarily characterized by recurrent painful mucocutaneous ulcerations. METHODS: A literature search was performed to write a narrative review into the pathogenesis and current treatment options of BD. RESULTS: The pathogenesis of BD remains to be elucidated, but is considered a genetically primed disease in which an external trigger causes immune activation resulting in inflammatory symptoms. GWAS data show an association between multiple genetic polymorphisms (HLA-B51, ERAP1, IL10 and IL23R-IL12RB2) and increased susceptibility to BD. Bacteria as streptococci, an unbalanced microbiome or molecular mimicry trigger the inflammation in BD. Increased production or responsiveness of pro-inflammatory components of the innate immune response (TLR, neutrophils, NK-cells or γδ T-cells) to these triggers may be a crucial step in the pathogenesis of BD. Additionally to an increased autoinflammatory response there is evidence of a dysregulated adaptive immune system, with a disturbed Th1/Th2 balance, expansion of Th17 cells and possibly a decrease in regulatory T cells, resulting in a surplus in pro-inflammatory cytokines. The inflammation causes a typical clinical phenotype including orogenital ulcerations, uveitis and skin lesions. Treatment is aimed at the aberrations found in the innate (neutrophils and γδ-T cells) and adaptive immune system (TNF-α, INF-γ, IL-1), directed at organ involvement and individualized based on patient characteristics. CONCLUSION: We presented an extensive review into the pathogenesis and treatment options of BD.

Therapeutic and biotechnological applications of substrate specific microbial aminopeptidases.

Aminopeptidases (EC 3.4.11.) belongs to exoprotease family, which can catalyze the cleavage of peptide bond which connects the N-terminal amino acid to the penultimate residue in a protein. Aminopeptidases catalyze the process of removal of the N-terminal amino acids of target substrates by sequential cleavage of one amino acid residue at a time. Microbial aminopeptidase are of great acceptance as industrial enzymes with varying applications in food and pharma industry since these enzymes possess unique characteristics than aminopeptidases from other sources. This review describes the various applications of microbial aminopeptidases in different industrial sectors. These enzymes are widely used in food industry as a debittering agent as well as in the preparation of protein hydrolysates. In baking, brewing, and cheese making aminopeptidases are extensively used for removing the bitterness of peptides. The inhibitors of these enzymes are found great clinical applications against various diseases such as cancer, diabetes, and viral infections. Aminopeptidases are widely used for the synthesis of biopeptides and amino acids, and found to be efficient than chemical synthesis. These enzymes are capable of hydrolyzing organophosphate compounds, thus having biological as well as environmental significance.Key Points• Cleaves the amino-terminal amino acid residues from proteins and peptides.• Microbial aminopeptidase are of great acceptance as both therapeutic and industrial enzyme.• Review describes the potential applications of microbial aminopeptidases.

Extraneuronal pathology in a canine model of CLN2 neuronal ceroid lipofuscinosis after intracerebroventricular gene therapy that delays neurological disease progression.

CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.

CLN2 Disease (Classic Late Infantile Neuronal Ceroid Lipofuscinosis).

CLN2 disease is an inherited metabolic storage disorder caused by the deficiency of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). The disease affects mainly the brain and the retina and is characterized by progressive dysfunction of the central nervous system, leading to dementia, epilepsy, loss of motor function and blindness. The classical late infantile type begins at around three years of age with epilepsy and/or a standstill of psychomotor development, followed by a rapid loss of all abilities and death in childhood. A late onset form in a small proportion of patients starts at the age of 4 to 10 years, but also leads to severe neurological deterioration. The deficiency of TPP1 causes the lysosomal accumulation of a material called ceroid lipofuscin. The natural substrate of TPP1 is not known, nor is the connection between storage process and neurodegeneration, which is characterized by loss of neurons. Among various experimental approaches to treatment, enzyme replacement therapy (ERT) and gene therapy have developed remarkably. Enzyme delivery through the cerebrospinal fluid led to wide distribution of enzyme activity in the brain and to attenuated neuropathology and disease progression in a TPP1-deficient mouse model as well as in a natural TPP1-deficient dog model. Safety of the intrathecal delivery, pharmacokinetics, and tissue distribution of the administered enzyme studied in non-human primates were encouraging, and a phase I/II clinical trial for intraventricular ERT in CLN2 patients is ongoing. A second approach uses intracerebral injection of viral vectors containing normal coding segments of the CLN2 gene. In a CLN2 mouse model, this procedure resulted in cerebral enzyme expression, reduced brain pathology and increased survival. A small number of patients have been treated the same way using an AAV2-vector for gene transfer to the brain. Although there were no serious adverse events unequivocally attributable to the vector used, there were some serious adverse effects, and a clinical benefit was not clearly evident under the conditions of the experiment. A phase I/phase II study using a AAVrh10 vector is presently recruiting patients.

Intracerebroventricular gene therapy that delays neurological disease progression is associated with selective preservation of retinal ganglion cells in a canine model of CLN2 disease.

CLN2 disease is one of a group of lysosomal storage disorders called the neuronal ceroid lipofuscinoses (NCLs). The disease results from mutations in the TPP1 gene that cause an insufficiency or complete lack of the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). TPP1 is involved in lysosomal protein degradation, and lack of this enzyme results in the accumulation of protein-rich autofluorescent lysosomal storage bodies in numerous cell types including neurons throughout the central nervous system and the retina. CLN2 disease is characterized primarily by progressive loss of neurological functions and vision as well as generalized neurodegeneration and retinal degeneration. In children the progressive loss of neurological functions typically results in death by the early teenage years. A Dachshund model of CLN2 disease with a null mutation in TPP1 closely recapitulates the human disorder with a progression from disease onset at approximately 4 months of age to end-stage at 10-11 months. Delivery of functional TPP1 to the cerebrospinal fluid (CSF), either by periodic infusion of the recombinant protein or by a single administration of a TPP1 gene therapy vector to the CSF, significantly delays the onset and progression of neurological signs and prolongs life span but does not prevent the loss of vision or modest retinal degeneration that occurs by 11 months of age. In this study we found that in dogs that received the CSF gene therapy treatment, the degeneration of the retina and loss of retinal function continued to progress during the prolonged life spans of the treated dogs. Eventually the normal cell layers of the retina almost completely disappeared. An exception was the ganglion cell layer. In affected dogs that received TPP1 gene therapy to the CSF and survived an average of 80 weeks, ganglion cell axons were present in numbers comparable to those of normal Dachshunds of similar age. The selective preservation of the retinal ganglion cells suggests that while TPP1 protein delivered via the CSF may protect these cells, preservation of the remainder of the retina will require delivery of normal TPP1 more directly to the retina, probably via the vitreous body.

Multifocal retinopathy in Dachshunds with CLN2 neuronal ceroid lipofuscinosis.

The CLN2 form of neuronal ceroid lipofuscinosis is an autosomal recessively inherited lysosomal storage disease that is characterized by progressive vision loss culminating in blindness, cognitive and motor decline, neurodegeneration, and premature death. CLN2 disease results from mutations in the gene that encodes the soluble lysosomal enzyme tripeptidyl peptidase-1. A null mutation in the TPP1 gene encoding this enzyme causes a CLN2-like disease in Dachshunds. Dachshunds that are homozygous for this mutation serve as a model for human CLN2 disease, exhibiting clinical signs and neuropathology similar to those of children with this disorder. Affected dogs reach end-stage terminal disease status at 10-11 months of age. In addition to retinal changes typical of CLN2 disease, a retinopathy consisting of multifocal, bullous retinal detachment lesions was identified in 65% of (TPP1-/-) dogs in an established research colony. These lesions did not occur in littermates that were heterozygous or homozygous for the normal TPP1 allele. Retinal changes and the functional effects of this multifocal retinopathy were examined objectively over time using ophthalmic examinations, fundus photography, electroretinography (ERG), quantitative pupillary light response (PLR) recording, fluorescein angiography, optical coherence tomography (OCT) and histopathology. The retinopathy consisted of progressive multifocal serous retinal detachments. The severity of the disease-related retinal thinning was no more serious in most detached areas than in adjacent areas of the retina that remained in close apposition to the retinal pigment epithelium. The retinopathy observed in these dogs was somewhat similar to canine multifocal retinopathy (CMR), a disease caused by a mutation of the bestrophin gene BEST1. ERG a-wave amplitudes were relatively preserved in the Dachshunds with CLN2 disease, whether or not they developed the multifocal retinopathy. The retinopathy also had minimal effects on the PLR. Histological evaluation indicated that the CLN2 disease-related retinal degeneration was not exacerbated in areas where the retina was detached except where the detached areas were very large. DNA sequence analysis ruled out a mutation in the BEST1 exons or splice junctions as a cause for the retinopathy. Perfect concordance between the TPP1 mutation and the retinopathy in the large number of dogs examined indicates that the retinopathy most likely occurs as a direct result of the TPP1 mutation. Therefore, inhibition of the development and progression of these lesions can be used as an indicator of the efficacy of therapeutic interventions currently under investigation for the treatment of CLN2 disease in the Dachshund model. In addition, these findings suggest that TPP1 mutations may underlie multifocal retinopathies of unknown cause in animals and humans.

Enzyme replacement therapy attenuates disease progression in a canine model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease).

Using a canine model of classical late-infantile neuronal ceroid lipofuscinosis (CLN2 disease), a study was conducted to evaluate the potential pharmacological activity of recombinant human tripeptidyl peptidase-1 (rhTPP1) enzyme replacement therapy administered directly to the cerebrospinal fluid (CSF). CLN2 disease is a hereditary neurodegenerative disorder resulting from mutations in CLN2, which encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Infants with mutations in both CLN2 alleles develop normally but in the late-infantile/early-childhood period undergo progressive neurological decline accompanied by pronounced brain atrophy. The disorder, a form of Batten disease, is uniformly fatal, with clinical signs starting between 2 and 4 years of age and death usually occurring by the early teenage years. Dachshunds homozygous for a null mutation in the canine ortholog of CLN2 (TPP1) exhibit a similar disorder that progresses to end stage at 10.5-11 months of age. Administration of rhTPP1 via infusion into the CSF every other week, starting at approximately 2.5 months of age, resulted in dose-dependent significant delays in disease progression, as measured by delayed onset of neurologic deficits, improved performance on a cognitive function test, reduced brain atrophy, and increased life span. Based on these findings, a clinical study evaluating the potential therapeutic value of rhTPP1 administration into the CSF of children with CLN2 disease has been initiated.

Enzyme replacement therapy delays pupillary light reflex deficits in a canine model of late infantile neuronal ceroid lipofuscinosis.

Late-infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a hereditary neurological disorder characterized by progressive retinal degeneration and vision loss, cognitive and motor decline, seizures, and pronounced brain atrophy. This fatal pediatric disease is caused by mutations in the CLN2 gene which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Utilizing a TPP1-/- Dachshund model of CLN2 disease, studies were conducted to assess the effects of TPP1 enzyme replacement administered directly to the CNS on disease progression. Recombinant human TPP1 (rhTPP1) or artificial cerebrospinal fluid vehicle was administered to CLN2-affected dogs via infusion into the CSF. Untreated and vehicle treated affected dogs exhibited progressive declines in pupillary light reflexes (PLRs) and electroretinographic (ERG) responses to light stimuli. Studies were undertaken to determine whether CSF administration of rhTPP1 alters progression of the PLR and ERG deficits in the canine model. rhTPP1 administration did not inhibit the decline in ERG responses, as rhTPP1 treated, vehicle treated, and untreated dogs all exhibited similar progressive and profound declines in ERG amplitudes. However, in some of the dogs treated with rhTPP1 there were substantial delays in the appearance and progression of PLR deficits compared with untreated or vehicle treated affected dogs. These findings indicate that CSF administration of TPP1 can attenuate functional impairment of neural pathways involved in mediating the PLR but does not prevent loss of retinal responses detectable with ERG.

Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: safety, pharmacokinetics, and distribution.

CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3-14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS.

Animal models for lysosomal storage disorders.

The lysosomal storage disorders (LSD) represent a heterogeneous group of inherited diseases characterized by the accumulation of non-metabolized macromolecules (by-products of cellular turnover) in different tissues and organs. LSDs primarily develop as a consequence of a deficiency in a lysosomal hydrolase or its co-factor. The majority of these enzymes are glycosidases and sulfatases, which in normal conditions participate in degradation of glycoconjugates: glycoproteins, glycosaminoproteoglycans, and glycolipids. Significant insights have been gained from studies of animal models, both in understanding mechanisms of disease and in establishing proof of therapeutic concept. These studies have led to the introduction of therapy for certain LSD subtypes, primarily by enzyme replacement or substrate reduction therapy. Animal models have been useful in elucidating molecular changes, particularly prior to onset of symptoms. On the other hand, it should be noted certain animal (mouse) models may have the underlying biochemical defect, but not show the course of disease observed in human patients. There is interest in examining therapeutic options in the larger spontaneous animal models that may more closely mimic the brain size and pathology of humans. This review will highlight lessons learned from studies of animal models of disease, drawing primarily from publications in 2011-2012.

New insights into the role of aminopeptidases in the treatment for both preeclampsia and preterm labor.

INTRODUCTION: Evidence elucidating the pathophysiology and pharmacology of conventional drugs, ß-2 stimulants and magnesium sulfate, on safety and effectiveness for preeclampsia and preterm labor are rarely found. Both compounds pass through the placental barrier and could exert their adverse effects on the fetus. Exposure to these agents could be problematic long after the birth, and possibly result in diseases such as autism and cardiomyopathy. Since 1970 the possible roles of placental aminopeptidases, which degrade peptide hormones, in preeclampsia and preterm labor have been studied. AREAS COVERED: Many studies reveal that the fetus secretes peptide hormones, such as angiotensin II, vasopressin, and oxytocin, under hypoxia (stress) during the course of its growth, suggesting the critical effects these hormones have during pregnancy. The roles of placental aminopeptidases, the enzymes which degrade fetal hormones without passing through the placental barrier, were clarified. A first-step production system for recombinant aminopeptidases was established, by which engineered recombinant aminopeptidases were used for further experiments testing expected efficacy on controlling the level of hormones. EXPERT OPINION: The authors conclude that both aminopeptidase A and placental leucine aminopeptidase could be potentially safe and effective drugs for patients and their babies in the treatment of preeclampsia and preterm labor.

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ∼10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.

Large-volume intrathecal enzyme delivery increases survival of a mouse model of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative lysosomal storage disorder caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl-peptidase (TPP1). LINCL primarily affects children, is fatal and there is no effective treatment. Administration of recombinant protein has proved effective in treatment of visceral manifestations of other lysosomal storage disorders but to date, only marginal improvement in survival has been obtained for neurological diseases. In this study, we have developed and optimized a large-volume intrathecal administration strategy to deliver therapeutic amounts of TPP1 to the central nervous system (CNS) of a mouse model of LINCL. To determine the efficacy of treatment, we have monitored survival as the primary endpoint and demonstrate that an acute treatment regimen (three consecutive daily doses started at 4 weeks of age) increases median lifespan of the LINCL mice from 16 (vehicle treated) to 23 weeks (enzyme treated). Consistent with the increase in life-span, we also observed significant reversal of pathology and improvement in neurological phenotype. These results provide a strong basis for both clinical investigation of large-volume/high-dose delivery of TPP1 to the brain via the cerebrospinal fluid (CSF) and extension of this approach towards other neurological lysosomal storage diseases.