Pharmacokinetics and Bioavailability of a Therapeutic Enzyme (Idursulfase) in Cynomolgus Monkeys after Intrathecal and Intravenous Administration

Intravenous enzyme replacement therapy with iduronate-2-sulfatase is an approved treatment for Hunter syndrome, however, conventional intravenous delivery cannot treat the neurologic manifestations of the disease due to its limited central nervous system penetration. Intrathecal administration of iduronate-2-sulfatase for delivery to the central nervous system is currently under investigation. The objective of this study was to evaluate the pharmacokinetics of idursulfase in the central nervous system of cynomolgus monkeys (Macaca fasicularis) after intravenous and intrathecal administration. Twenty-seven monkeys, treatment-naïve to enzyme replacement therapy, were placed into 4 groups according to body weight: Group 1 was administered 0.5 mg/kg idursulfase intravenously, Groups 2–4 were administered an intrathecal formulation (1-, 10-, and 30-mg doses). Blood samples and cerebrospinal fluid (sampled at the cisterna magna or lumbar level) were collected at the same time points for 72 hours post dosing. Following intravenous administration, a high maximum serum concentration and rapid distribution of iduronate-2-sulfatase out of the central compartment were observed (elimination half-life: 4.3 hours). Iduronate-2-sulfatase exposure in the cerebrospinal fluid was limited, suggesting intravenous administration provided minimal penetration of the blood–brain barrier. Following intrathecal administration, a high maximum observed concentration was immediately noted and elimination half-life ranged between 7.8–10 hours and 5.9–6.7 hours (cisterna magna and lumbar sampling, respectively). Cerebrospinal fluid pharmacokinetic profiles at different doses of iduronate-2-sulfatase were similar and the dose/exposure relationship was proportional. After intrathecal administration, movement of iduronate-2-sulfatase from cerebrospinal fluid to serum was observed (systemic bioavailability was 40–83%). The clear penetration of iduronate-2-sulfatase into the cerebrospinal fluid and the dose response suggest that intrathecal delivery of iduronate-2-sulfatase may be suitable for treating the central nervous system manifestations associated with Hunter syndrome.     

IDS Crossing of the Blood-Brain Barrier Corrects CNS Defects in MPSII Mice

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, arises from a deficiency in iduronate 2-sulfatase (IDS), and it is characterized by progressive somatic and neurological involvement. The MPSII mouse model reproduces the features of MPSII patients. Systemic administration of the AAV2/5CMV-hIDS vector in MPSII mouse pups results in the full correction of glycosaminoglycan (GAG) accumulation in visceral organs and in the rescue of the defects and GAG accumulation in the central nervous system (CNS). Remarkably, in treated MPSII animals, this CNS correction arises from the crossing of the blood-brain barrier by the IDS enzyme itself, not from the brain transduction. Thus, we show here that early treatment of MPSII mice with one systemic injection of AAV2/5CMV-hIDS results in prolonged and high levels of circulating IDS that can efficiently and simultaneously rescue both visceral and CNS defects for up to 18 months after therapy.     

Peripheral Nervous System Neuropathology and Progressive Sensory Impairments in a Mouse Model of Mucopolysaccharidosis IIIB

The lysosomal storage pathology in Mucopolysaccharidosis (MPS) IIIB manifests in cells of virtually all organs. However, it is the profound role of the neurological pathology that leads to morbidity and mortality in this disease, and has been the major challenge to developing therapies. To date, MPS IIIB neuropathologic and therapeutic studies have focused predominantly on changes in the central nervous system (CNS), especially in the brain, and little is known about the disease pathology in the peripheral nervous system (PNS). This study demonstrates characteristic lysosomal storage pathology in dorsal root ganglia affecting neurons, satellite cells (glia) and Schwann cells. Lysosomal storage lesions were also observed in the myoenteric plexus and submucosal plexus, involving enteric neurons with enteric glial activation. Further, MPS IIIB mice developed progressive impairments in sensory functions, with significantly reduced response to pain stimulation that became detectable at 4–5 months of age as the disease progressed. These data demonstrate that MPS IIIB neuropathology manifests not only in the entire CNS but also the PNS, likely affecting both afferent and efferent neural signal transduction. This study also suggests that therapeutic development for MPS IIIB may benefit from targeting the entire nervous system.     

Guidelines for diagnosis and treatment of Hunter Syndrome for clinicians in Latin America

This review aims to provide clinicians in Latin America with the most current information on the clinical aspects, diagnosis, and management of Hunter syndrome, a serious and progressive disease for which specific treatment is available. Hunter syndrome is a genetic disorder where iduronate-2-sulfatase (I2S), an enzyme that degrades glycosaminoglycans, is absent or deficient. Clinical manifestations vary widely in severity and involve multiple organs and tissues. An attenuated and a severe phenotype are recognized depending on the degree of cognitive impairment. Early diagnosis is vital for disease management. Clinical signs common to children with Hunter syndrome include inguinal hernia, frequent ear and respiratory infections, facial dysmorphisms, macrocephaly, bone dysplasia, short stature, sleep apnea, and behavior problems. Diagnosis is based on screening urinary glycosaminoglycans and confirmation by measuring I2S activity and analyzing I2S gene mutations. Idursulfase (recombinant I2S) (Elaprase^®, Shire) enzyme replacement therapy (ERT), designed to address the underlying enzyme deficiency, is approved treatment and improves walking capacity and respiratory function, and reduces spleen and liver size and urinary glycosaminoglycan levels. Additional measures, responding to the multi-organ manifestations, such as abdominal/inguinal hernia repair, carpal tunnel surgery, and cardiac valve replacement, should also be considered. Investigational treatment options such as intrathecal ERT are active areas of research, and bone marrow transplantation is in clinical practice. Communication among care providers, social workers, patients and families is essential to inform and guide their decisions, establish realistic expectations, and assess patients’ responses.     

Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases.     

In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors.

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme alpha-L-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

The effect of four mutations on the expression of iduronate-2-sulfatase in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13). Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named DeltaS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype-phenotype correlation, prognosis and possible therapeutic intervention.     

Intrathecal Fas ligand infusion strengthens immunoprivilege of central nervous system and suppresses experimental autoimmune encephalomyelitis

Fas ligand (FasL) is an essential molecule strongly expressed in some immunoprivileged sites, but is expressed at very low levels in normal CNS. In this study, acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats with guinea pig myelin basic protein. Intrathecal infusion of recombinant FasL before EAE onset dose dependently suppressed acute EAE and alleviated pathological inflammation in lumbosacral spinal cord. This treatment greatly increased apoptosis in CNS inflammatory cells, but did not inhibit systemic immune response to myelin basic protein. Systemic administration of a similar dose of rFasL was ineffective. In vitro, encephalitogenic T cells were highly sensitive to rFasL-induced cell death, and activated macrophages were also susceptible. In addition, in vitro rFasL treatment potentiated the immunosuppressive property of rat cerebrospinal fluid. We conclude that intrathecal infusion of rFasL eliminated the initial wave of infiltrating T cells and macrophages, and therefore blocked the later recruitment of inflammatory cells into CNS. Although Fas receptor expression was observed on spinal cord neurons, astrocytes, and oligodendrocytes, no damage to these cells or to the myelin structure was detected after rFasL infusion.     

Delivery,Distribution, and Neuronal Uptake of Exogenous Mannose-Terminal Glucocerebrosidase in the Intact Rat Brain

Gaucher disease is caused by insufficient activity of the enzyme glucocerebrosidase. Great benefit has been obtained through enzyme replacement therapy for patients with type 1 (non-neuronopathic) Gaucher disease. In contrast, inconsistent effects of enzyme therapy have been observed in patients with type 3 (chronic neuronopathic) Gaucher disease, and no benefit on the lethal course of the disease occurs in patients with Type 2 (acute neuronopathic) Gaucher disease. We examined the use of convection-enhanced delivery to augment the delivery and distribution of exogenous glucocerebrosidase (m.w. 63,000) to the brain by infusing it under slight hydrostatic pressure into the striatal region of rats. The enzyme was comparatively stable under these conditions. It was distributed from the site of injection toward the cerebral cortex where it became primarily localized in neurons. These findings provide considerable incentive for the exploration of intracerebral microinfusion of enzyme to the brain of patients with metabolic storage disorders involving the CNS.     

Recombinant AAV-mediated gene delivery to the central nervous system

Various regions of the brain have been successfully transduced by recombinant adeno-associated virus (rAAV) vectors with no detected toxicity. When using the cytomegalovirus immediate early (CMV) promoter, a gradual decline in the number of transduced cells has been described. In contrast, the use of cellular promoters such as the neuron-specific enolase promoter or hybrid promoters such as the chicken beta-actin/CMV promoter resulted in sustained transgene expression. The cellular tropism of rAAV-mediated gene transfer in the central nervous system (CNS) varies depending on the serotype used. Serotype 2 vectors preferentially transduce neurons whereas rAAV5 and rAAV1 transduce both neurons and glial cells. Recombinant AAV4-mediated gene transfer was inefficient in neurons and glial cells of the striatum (the only structure tested so far) but efficient in ependymal cells. No inflammatory response has been described following rAAV2 administration to the brain. In contrast, antibodies to AAV2 capsid and transgene product were elicited but no reduction of transgene expression was observed and readministration of vector without loss of efficiency was possible from 3 months after the first injection. Based on the success of pioneer work performed with marker genes, various strategies for therapeutic gene delivery were designed. These include enzyme replacement in lysosomal storage diseases, Canavan disease and Parkinson's disease; delivery of neuroprotective factors in Parkinson's disease, Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and spinal cord injury; as well as modulation of neurotransmission in epilepsy and Parkinson's disease. Several of these strategies have demonstrated promising results in relevant animal models. However, their implementation in the clinics will probably require a tight regulation and a specific targeting of therapeutic gene expression which still demands further developments of the vectors.     

Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture

We have used antibody and complement-mediated cell killing, fluorescence-activated cell sorting and tissue culture to study the development of rat oligodendrocytes. We show that (1) three ligands that bind to the majority of CNS neurons (the monoclonal antibodies A4 and A2B5 and tetanus toxin) also bind to immature oligodendrocytes and to precursor cells in 14-day embryonic rat brain that develop into oligodendrocytes in vitro; and (2) precursor cells in 17- to 18-day embryonic rat optic nerve can develop into oligodendrocytes in vitro in the absence of living neurons.     

Testosterone metabolism in brain cells and membranes

The central nervous system (CNS) is considered a target structure for the action of all the classes of hormonal steroids produced by the organism. Well-characterized genomic and less well-understood membrane mechanisms of action are probably involved in the steroid modulation of brain activities. Moreover, some classes of steroids need to be converted into “active” metabolites before interacting with their effector systems. In particular, testosterone (T) exerts many of its effects after conversion to 5-dihydrotestosterone (DHT) and estrogens. The CNS possesses both the 5-reductase, the enzyme which produces DHT and the aromatase which transforms T into estrogens; however, the relative role and distribution of these enzymes in the various structural components of the CNS has not been clarified so far. The 5-reductase has been found to be present in high concentrations in brain white matter structures because these are particularly rich in myelin membranes, to which the enzymatic activity appears to be associated. This membrane localization might suggest a possible involvement of steroidal 5-reduced metabolites in membrane-mediated events in the CNS. Moreover, the distribution of 5-reductase was studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation, as well as in neurons and glial cells grown in culture. The aromatase activity was also evaluated in neurons and glial cells grown in culture and in isolated oligodendrocytes. Among the three cell types isolated, neurons appear to be more active than oligodendrocytes and astrocytes, respectively, in converting T into DHT. Also, in cell culture experiments, neurons are more active in forming DHT than glial cells. Only neurons possess aromatase activity, while glial cells are apparently unable to aromatize T.     

Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier

Mucopolysaccharidosis Type I, Hurler's Syndrome, is a lysosomal storage disorder that affects the brain. The missing enzyme, alpha-L-iduronidase (IDUA), does not cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, human IDUA was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry into brain the IDUA. Transfection of COS cells resulted in high levels of IDUA enzyme activity both in the medium and in the intracellular space. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDUA or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The IDUA enzyme specific activity of the affinity purified HIRMAb-IDUA fusion protein was 363 +/- 37 U/microg protein, which is comparable to specific activity of recombinant IDUA. The accumulation of glycosoaminoglycans in Hurler fibroblasts was decreased 70% by treatment with the HIRMAb-IDUA fusion protein. Confocal microscopy showed targeting of the fusion protein to the lysosome. The HIRMAb-IDUA fusion protein bound with high affinity to the HIR, and was rapidly transported into the brain of the adult Rhesus monkey following intravenous administration. The HIRMAb-IDUA fusion protein is a new treatment for Hurler's syndrome, which has been specifically engineered to cross the human BBB.     

Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

Neuropathic pain is a debilitating pain condition that occurs after nerve damage. Such pain is considered to be a reflection of the aberrant excitability of dorsal horn neurons. Emerging lines of evidence indicate that spinal microglia play a crucial role in neuronal excitability and the pathogenesis of neuropathic pain, but the mechanisms underlying neuron-microglia communications in the dorsal horn remain to be fully elucidated. A recent study has demonstrated that platelet-derived growth factor (PDGF) expressed in dorsal horn neurons contributes to neuropathic pain after nerve injury, yet how PDGF produces pain hypersensitivity remains unknown. Here we report an involvement of spinal microglia in PDGF-induced tactile allodynia. A single intrathecal delivery of PDGF B-chain homodimer (PDGF-BB) to naive rats produced a robust and long-lasting decrease in paw withdrawal threshold in a dose-dependent manner. Following PDGF administration, the immunofluorescence for phosphorylated PDGF β-receptor (p-PDGFRβ), an activated form, was markedly increased in the spinal dorsal horn. Interestingly, almost all p-PDGFRβ-positive cells were double-labeled with an antibody for the microglia marker OX-42, but not with antibodies for other markers of neurons, astrocytes and oligodendrocytes. PDGF-stimulated microglia in vivo transformed into a modest activated state in terms of their cell number and morphology. Furthermore, PDGF-BB-induced tactile allodynia was prevented by a daily intrathecal administration of minocycline, which is known to inhibit microglia activation. Moreover, in rats with an injury to the fifth lumbar spinal nerve (an animal model of neuropathic pain), the immunofluorescence for p-PDGFRβ was markedly enhanced exclusively in microglia in the ipsilateral dorsal horn. Together, our findings suggest that spinal microglia critically contribute to PDGF-induced tactile allodynia, and it is also assumed that microglial PDGF signaling may have a role in the pathogenesis of neuropathic pain.     

Sympathoexcitation to intravenous interleukin-1beta is dependent on forebrain neural circuits

We investigated the contributions of forebrain, brain stem, and spinal neural circuits to interleukin (IL)-1beta-induced sympathetic nerve discharge (SND) responses in alpha-chloralose-anesthetized rats. Lumbar and splenic SND responses were determined in spinal cord-transected (first cervical vertebra, C1), midbrain-transected (superior colliculus), and sham-transected rats before and for 60 min after intravenous IL-1beta (285 ng/kg). The observations made were the following: 1) lumbar and splenic SND were significantly increased after IL-1beta in sham C1-transected rats but were unchanged after IL-1beta in C1-transected rats; 2) intrathecal administration of DL-homocysteic acid (10 ng) increased SND in C1-transected rats; 3) lumbar and splenic SND were significantly increased after IL-1beta in sham- but not midbrain-transected rats; and 4) midbrain transection did not alter the pattern of lumbar and splenic SND, demonstrating the integrity of brain stem sympathetic neural circuits after decerebration. These results demonstrate that an intact forebrain is required for mediating lumbar and splenic sympathoexcitatory responses to intravenous IL-1beta, thereby providing new information about the organization of neural circuits responsible for mediating sympathetic-immune interactions.     

The cellular and molecular events of central nervous system remyelination

Central nervous system (CNS)* regeneration is a subject of great interest, particularly in diseases causing a dramatic loss of neurons. However, some CNS diseases do not affect neurons but damage other cells, such as the myelin-forming cells--called oligodendrocytes--which are also crucial to the harmonious function of the nervous system. Diseases in which oligodendrocytes and myelin are attacked can cause devastating neurological dysfunction which is sometimes followed by recovery and myelin repair or remyelination. The question of the regeneration potential of oligodendrocytes in experimental and human demyelinating diseases such as multiple sclerosis has been debated for a long time. Present evidence suggests that oligodendrocyte precursor cells persist in the adult CNS and that oligodendrocyte regeneration can occur but may be limited by ongoing disease processes. Here we will briefly review recent advances which have broadened our understanding of the cellular and molecular events of CNS remyelination.     

Human α-N-acetylglucosaminidase: cDNA cryptic site removal and native secretion signal addition significantly enhance enzyme expression and secretion

Human α-N-acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in Mucopolysaccharidosis type IIIB (MPS IIIB). We utilize a Spodoptera frugiperda (Sf9) system to express Naglu fused to a synthetic protein transduction domain in hopes to facilitate delivery of Naglu across the blood–brain barrier, thus allowing enzyme replacement therapy to treat neurological symptoms.Although human recombinant Naglu was previously produced in Sf9, low expression levels suggested degradation via cryptic mRNA splicing. Two cryptic splice sites discovered within Naglu cDNA were altered by site-directed mutagenesis, reducing Naglu mRNA degradation. A native Naglu secretion-signaling peptide was efficiently recognized by the Sf9 system. Significantly higher enzyme activity was seen from multiple adherent Sf9 cultures stably expressing mutagenized Naglu over those expressing wildtype Naglu (P = 0.000; 3.4-fold average increased specific activity). Suspension cultures demonstrated a 4.0-fold increase in overall enzyme activity secreted post-mutagenesis. Thus elimination of cryptic splicing directly resulted in higher Naglu expression.