The Differences of Mechanisms in Antihypertensive and Anti-Obesity Effects of Eucommia Leaf Extract between Rodents and Humans.

In the 1970s, Eucommia leaf tea, known as Tochu-cha in Japanese, was developed from roasted Eucommia leaves in Japan and is considered as a healthy tea. The antihypertensive, diuretic, anti-stress, insulin resistance improving, and anti-obesity effects of Eucommia leaf extract have been reported. However, the identification and properties of the active components as well as the underlying mechanism of action are largely unknown. In this review, we summarize studies involving the oral administration of geniposidic acid, a major iridoid component of Eucommia leaf extract which increases plasma atrial natriuretic peptide (ANP) on the atria of spontaneously hypertensive rats (SHR) by activating the glucagon-like peptide-1 receptor (GLP-1R). To achieve the antihypertensive effects of the Eucommia leaf extract through ANP secretion in humans, combining a potent cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) inhibitor, such as pinoresinol di-ß-d-glucoside, with geniposidic acid may be necessary. Changes in the gut microbiota are an important aspect involved in the efficacy of asperuloside, another component of the Eucommia leaf extract, which improves obesity and related sequelae, such as insulin resistance and glucose intolerance. There are species differences of mechanisms associated with the antihypertensive and anti-obesity effects between rodents and humans, and not all animal test results are consistent with that of human studies. This review is focused on the mechanisms in antihypertensive and anti-obesity effects of the Eucommia leaf extract and summarizes the differences of mechanisms in their effects on rodents and humans based on our studies and those of others.

Eucommia Leaf Extract Induces BDNF Production in Rat Hypothalamus and Enhances Lipid Metabolism and Aerobic Glycolysis in Rat Liver.

BACKGROUND: Mutations in the brain-derived neurotrophic factor (BDNF) gene and its receptor, tyrosine receptor kinase B (TrkB), have been reported to cause severe obesity in rodents. Our previous study demonstrated that the oral administration of 5% Eucommia leaf extract (ELE) or ELE aroma treatment (ELE aroma) produced anti-obesity effects. OBJECTIVE: In this study, we investigated the effects of ELE on glycolysis and lipid metabolism in male Sprague-Dawley rats, as well as the effects of ELE on BDNF in rat hypothalamus. METHODS AND RESULTS: A significant reduction and a reduction tendency in the respiratory quotient were observed in association with 5% ELE and ELE aroma treatment, respectively. Furthermore, RT-qPCR results showed significant increases in Cpt2, Acad, Complex II, and Complex V mRNA levels in the liver with both treatments. In addition, in rat hypothalamus, significant elevations in BDNF, Akt, PLCγ proteins and CREB phosphorylation were observed in the 5% ELE group and the ELE aroma group. Furthermore, the Ras protein was significantly increased in the ELE aroma group. On the other hand, significant dephosphorylation of ERK1/2 was observed by the western blotting in the 5% ELE group and the ELE aroma group. CONCLUSION: These findings suggest that the ELE treatment enhances the lipid metabolism and increases the aerobic glycolytic pathway, while ELE-induced BDNF may affect such energy regulation. Therefore, ELE has the possibility to control metabolic syndrome.

Combination of syringaresinol-di-O-ß-D-glucoside and chlorogenic acid shows behavioral pharmacological anxiolytic activity and activation of hippocampal BDNF-TrkB signaling.

Mental stress, such as anxiety and conflict, causes physiological changes such as dysregulation of autonomic nervous activity, depression, and gastric ulcers. It also induces glucocorticoid production and changes in hippocampal brain-derived neurotrophic factor (BDNF) levels. We previously reported that Acanthopanax senticosus HARMS (ASH) exhibited anxiolytic activity. Thus, we attempted to identify the anxiolytic constituents of ASH and investigated its influence on hippocampal BDNF protein expression in male Sprague Dawley rats administered chlorogenic acid (CHA), ( +)-syringaresinol-di-O-ß-D-glucoside (SYG), or a mixture of both (Mix) for 1 week using the open field test (OFT) and improved elevated beam walking (IEBW) test. As with ASH and the benzodiazepine anxiolytic cloxazolam (CLO), Mix treatment significantly increased locomotor activity in the OFT. CHA and Mix increased the time spent in the open arm in the IEBW test. SYG and Mix treatment inhibited the significant increase in normalized low-frequency power, indicative of sympathetic nervous activity, and significant decrease in normalized high-frequency power, indicative of parasympathetic nervous activity, as observed in the IEBW test. SYG and Mix treatment significantly increased hippocampal BDNF protein expression. The combination of CHA and SYG possibly induces anxiolytic behavior and modulates autonomic regulation, activates hippocampal BDNF signaling as with ASH.

Tranexamic Acid Protects Ovary and Testis Functions and Ameliorates Osteoporosis in Mice.

INTRODUCTION: In a rapidly aging society, the number of people suffering from osteoporosis keeps increasing. However, effective prevention strategies for osteoporosis are not yet currently available. OBJECTIVE: In this study, we examined the ameliorative effects of tranexamic acid on osteoporosis in 24-month-old mice. METHODS: During the study period, mice were orally administered tranexamic acid 3 times per week. RESULTS: Bone mineral density, which is a parameter of osteoporosis, was improved following tranexamic acid administration. In addition, female mice evidenced a stronger phenotypic improvement than male mice. In female mice treated with tranexamic acid, ovary abnormalities were reduced. Furthermore, the levels of transforming growth factor-ß, hyaluronic acid, CD44, reactive oxygen species, and apoptosis, as well as the number of infiltrated neutrophils and macrophages in the ovary were lower than those in the control or solvent-administered mice. In addition, 17ß-estradiol levels in blood increased when compared with the control or solvent-treated mice. In addition, administration of tranexamic acid to 24-month-old male mice decreased the level of apoptosis in the testis. However, the levels of 17ß-estradiol and testosterone in blood increased compared with the control or solvent-administered mice. CONCLUSIONS: The use of tranexamic acid had an ameliorative effect on osteoporosis, possibly by protecting ovaries and testes.

Anxiolytic Effects of Acanthopanax senticosus HARMS Occur via Regulation of Autonomic Function and Activate Hippocampal BDNF⁻TrkB Signaling.

Mental stress, such as anxiety and conflict, causes physiological changes, such as changes in autonomic nervous activity and gastric ulcers. In addition, stress induces glucocorticoids and changes the hippocampal brain-derived neurotrophic factor (BDNF) expression levels. We previously reported that Acanthopanax senticosus HARM (ASH) prevents stress-induced gastric ulcers. Thus, we investigated the potential anxiolytic effect and influence of ASH on the hippocampus BDNF-related protein in male Sprague-Dawley rats fed 1% and 5% ASH extract-containing food for one week using novelty suppressed feeding (NSF) and improved elevated beam walking (IEBW) tests. ASH treatment significantly decreased latency to eat in the NSF test and increased the time spent on the open arm in the IEBW test. ASH5% treatment showed a significant decrease in LFnu, indicative of sympathetic nervous activity, and a significant increase in HFnu, indicative of parasympathetic nervous activity, in the NSF test. In addition, ASH1% and ASH5% treatments significantly decreased LFnu and significantly increased HFnu in the IEBW test. ASH5% treatment significantly increased hippocampal BDNF protein expression in both Western blotting and immunohistochemistry experiments. Our findings suggest that anxiolytic effects of ASH occur via the regulation of autonomic function and increased hippocampal BDNF signaling.

Valproic acid mediates miR-124 to down-regulate a novel protein target, GNAI1.

Valproic acid (VPA) is an anti-convulsant drug that is recently shown to have neuroregenerative therapeutic actions. In this study, we investigate the underlying molecular mechanism of VPA and its effects on Bdnf transcription through microRNAs (miRNAs) and their corresponding target proteins. Using in silico algorithms, we predicted from our miRNA microarray and iTRAQ data that miR-124 is likely to target at guanine nucleotide binding protein alpha inhibitor 1 (GNAI1), an adenylate cyclase inhibitor. With the reduction of GNAI1 mediated by VPA, the cAMP is enhanced to increase Bdnf expression. The levels of GNAI1 protein and Bdnf mRNA can be manipulated with either miR-124 mimic or inhibitor. In summary, we have identified a novel molecular mechanism of VPA that induces miR-124 to repress GNAI1. The implication of miR-124→GNAI1→BDNF pathway with valproic acid treatment suggests that we could repurpose an old drug, valproic acid, as a clinical application to elevate neurotrophin levels in treating neurodegenerative diseases.

Enzyme replacement therapy in newborn mucopolysaccharidosis IVA mice: early treatment rescues bone lesions?

We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology [1]. To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in the liver and spleen, with detectable activity in the bone and brain. Second, newborn ERT was conducted after a tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th weeks were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in the liver and spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than that in adult mice treated with ERT; however, hyaline and fibrous cartilage cells in the femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mice.

Enzyme replacement therapy on hypophosphatasia mouse model.

Hypophosphatasia (HPP) is an inborn error of metabolism caused by deficiency of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5-phosphate (PLP), a co-factor form of vitamin B6. Enzyme replacement therapy (ERT) for HPP by functional TNSALP is one of the therapeutic options. The C-terminal-anchorless human recombinant TNSALP derived from Chinese hamster ovary cell lines was purified. TNSALP-null mice (Akp2 (-/-) ), an infantile model of HPP, were treated from birth using TNSALP and vitamin B6 diet. Long-term efficacy studies of ERT consisted of every 3 days subcutaneous or intravenous injections till 28 days old (dose 20 U/g) and subsequently every 3 days intravenous injections for 6 months (dose 10 U/g). We assessed therapeutic effect by growth and survival rates, fertility, skeletal manifestations, and radiographic and pathological finding. Treated Akp2 (-/-) mice grew normally till 4 weeks and appeared well with a minimum skeletal abnormality as well as absence of epilepsy, compared with untreated mice which died by 3 weeks old. The prognosis of TNSALP-treated Akp2 (-/-) mice was improved substantially: 1) prolonged life span over 6 months, 2) improvement of the growth, and 3) normal fertility. After 6 months of treatment, we found moderate hypomineralization with abnormal proliferative chondrocytes in growth plate and articular cartilage. In conclusion, ERT with human native TNSALP improves substantial clinical manifestations in Akp2 (-/-) mice, suggesting that ERT with anchorless TNSALP is also a potential therapy for HPP.

Long circulating enzyme replacement therapy rescues bone pathology in mucopolysaccharidosis VII murine model.

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme ß-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

The role of miRNAs in complex formation and control.

UNLABELLED: microRibonucleic acid (miRNAs) are small regulatory molecules that act by mRNA degradation or via translational repression. Although many miRNAs are ubiquitously expressed, a small subset have differential expression patterns that may give rise to tissue-specific complexes. MOTIVATION: This work studies gene targeting patterns amongst miRNAs with differential expression profiles, and links this to control and regulation of protein complexes. RESULTS: We find that, when a pair of miRNAs are not expressed in the same tissues, there is a higher tendency for them to target the direct partners of the same hub proteins. At the same time, they also avoid targeting the same set of hub-spokes. Moreover, the complexes corresponding to these hub-spokes tend to be specific and nonoverlapping. This suggests that the effect of miRNAs on the formation of complexes is specific.

Tsunami lung.

We encountered three cases of lung disorders caused by drowning in the recent large tsunami that struck following the Great East Japan Earthquake. All three were females, and two of them were old elderly. All segments of both lungs were involved in all the three patients, necessitating ICU admission and endotracheal intubation and mechanical ventilation. All three died within 3 weeks. In at least two cases, misswallowing of oil was suspected from the features noted at the time of the detection. Sputum culture for bacteria yielded isolation of Stenotrophomonas maltophilia, Legionella pneumophila, Burkholderia cepacia, and Pseudomonas aeruginosa. The cause of tsunami lung may be a combination of chemical induced pneumonia and bacterial pneumonia.

1H-magnetic resonance spectroscopy indicates damage to cerebral white matter in the subacute phase after CO poisoning.

OBJECTIVE: The authors examined whether (1)H-magnetic resonance spectroscopy (MRS) can identify damage to the centrum semiovale in the subacute phase after CO exposure. METHODS: Subjects comprised 29 adult patients who were treated with hyperbaric oxygenation within a range of 4-95 h (mean 18.7 h) after CO exposure. Subjects were classified into three groups according to clinical behaviours: Group A, patients with transit acute symptoms only; Group P, patients with persistent neurological symptoms; and Group D, patients with 'delayed neuropsychiatric sequelae' occurring after a lucid interval. MRS of bilateral centrum semiovale was performed 2 weeks after CO inhalation for all patients and 13 healthy volunteers. The mean ratios of choline-containing compounds/creatine ((mean)Cho/Cr) and N-acetylaspartate/Cr ((mean)NAA/Cr) for bilateral centrum semiovale were calculated and compared between the three CO groups and controls. Myelin basic protein (MBP) concentration in cerebrospinal fluid was examined at 2 weeks to evaluate the degree of demyelination in patients. RESULTS: MBP concentration was abnormal for almost all patients in Groups P and D, but was not abnormal for any Group A patients. The (mean)Cho/Cr ratios were significantly higher in Groups P and D than in Group A. No significant difference in (mean)NAA/Cr ratio was seen between the three pathological groups and controls. A significant correlation was identified between MBP and (mean)Cho/Cr ratio. CONCLUSIONS: These results suggest that the Cho/Cr ratio in the subacute phase after CO intoxication represents early demyelination in the centrum semiovale, and can predict chronic neurological symptoms.

Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography-tandem mass spectrometry.

Mucopolysaccharidosis type IVA (MPS IVA, Morquio A disease), a progressive lysosomal storage disease, causes skeletal chondrodysplasia through excessive storage of keratan sulfate (KS). KS is synthesized mainly in cartilage and released to the circulation. The excess storage of KS disrupts cartilage, consequently releasing more KS into circulation, which is a critical biomarker for MPS IVA. Thus, assessment of KS level provides a potential screening strategy and determines clinical course and efficacy of therapies. We have recently developed a tandem mass spectrometry liquid chromatography [LC/MS/MS] method to assay KS levels in blood. Forty-nine blood specimens from patients with MPS IVA [severe (n = 33), attenuated (n = 11) and undefined (n = 5)] were analyzed for comparison of blood KS concentration with that of healthy subjects and for correlation with clinical severity. Plasma samples were digested by keratanase II to obtain disaccharides of KS. Digested samples were assayed by LC/MS/MS. We found that blood KS levels (0.4-26 µg/ml) in MPS IVA patients were significantly higher than those in age-matched controls (0.67-4.6 µg/ml; P < 0.0001). It was found that blood KS level varied with age and clinical severity in the patients. Blood KS levels in MPS IVA peaked between 2 years and 5 years of age (mean 11.4 µg/ml). Blood KS levels in severe MPS IVA (mean 7.3 µg/ml) were higher than in the attenuated form (mean 2.1 µg/ml) (P = 0.012). We also found elevated blood KS levels in other types of MPS. These findings indicate that the new KS assay for blood is suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Assessment of damage to cerebral white matter fiber in the subacute phase after carbon monoxide poisoning using fractional anisotropy in diffusion tensor imaging.

INTRODUCTION: Chronic neuropsychiatric symptoms after carbon monoxide (CO) poisoning are caused by demyelination of cerebral white matter fibers. We examined whether diffusion tensor imaging can sensitively represent damage to fibers of the centrum semiovale in the subacute phase after CO intoxication. METHODS: Subjects comprised 13 adult patients with CO poisoning, classified into three groups according to clinical behaviors: group A, patients with transit acute symptoms only; group P, patients with persistent neurological symptoms; and group D, patients with "delayed neuropsychiatric sequelae" occurring after a lucid interval. Median fractional anisotropy (FA) and apparent diffusion coefficient (ADC) of the centrum semiovale bilaterally at 2 weeks were compared between these groups and a control group of ten healthy volunteers. Myelin basic protein (MBP) concentration in cerebrospinal fluid was examined at 2 weeks to evaluate the degree of demyelination in patients. RESULTS: MBP concentration was abnormal or detectable for all group P and group D patients but was undetectable for all patients assigned to group A. Low FA values in groups P and D displaying chronic neurological symptoms clearly differed from those in controls and group A without chronic neurological symptoms, but ADC showed no significant differences between patient groups. CONCLUSIONS: MBP concentration at 2 weeks after CO inhalation confirmed a certain extent of demyelination in the central nervous system of patients who would develop chronic neurological symptoms. In these patients, FA sensitively represented damage to white matter fibers in the centrum semiovale in the subacute phase after CO intoxication.

Validation of disaccharide compositions derived from dermatan sulfate and heparan sulfate in mucopolysaccharidoses and mucolipidoses II and III by tandem mass spectrometry.

Glycosaminoglycans (GAGs) are accumulated in various organs in both mucopolysaccharidoses (MPS) and mucolipidoses II and III (ML II and III). MPS and ML II and III patients can not properly degrade dermatan sulfate (DS) and/or heparan sulfate (HS). HS storage occurs in the brain leading to neurological signs while DS storage involves mainly visceral and skeletal manifestations. Excessive DS and HS released into circulation and thus blood levels of both are elevated, therefore, DS and HS in blood could be critical biomarkers for MPS and ML. Such measurement can provide a potential early screening, assessment of the clinical course and efficacy of therapies. We here assay DS and HS levels in MPS and ML patients using liquid chromatography tandem mass spectrometry (LC/MS/MS). Plasma samples were digested by heparitinase and chondroitinase B to obtain disaccharides of DS and HS, followed by LC/MS/MS analysis. One hundred-twenty samples from patients and 112 control samples were analyzed. We found that all MPS I, II, III and VI patients had a significant elevation of all DS+HS compositions analyzed in plasma, compared with the controls (P<0.0001). Specificity and sensitivity was 100% if the cut off value is 800 ng/ml between control and these types of MPS group. All MPS I, II and III patients also had a significant elevation of plasma HS, compared with the controls (P<0.0001). All MPS VI patients had a significant elevation of plasma DS, compared with the controls (P<0.0001). These findings suggest measurement of DS and/or HS levels by LC/MS/MS is applicable to the screening for MPS I, II, III and VI patients.

Acidic amino acid tag enhances response to enzyme replacement in mucopolysaccharidosis type VII mice.

We have tested an acidic oligopeptide-based targeting system for delivery of enzymes to tissues, especially bone and brain, in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy is based upon tagging a short peptide consisting of acidic amino acids (AAA) to N terminus of human beta-glucuronidase (GUS). The pharmacokinetics, biodistribution, and the pathological effect on MPS VII mouse after 12 weekly infusions were determined for recombinant human untagged and tagged GUS. The tagged GUS was taken up by MPS VII fibroblasts in a mannose 6-phosphate receptor-dependent manner. Intravenously injected AAA-tagged enzyme had five times more prolonged blood clearance compared with the untagged enzyme. The tagged enzyme was delivered effectively to bone, bone marrow, and brain in MPS VII mice and was effective in reversing the storage pathology. The storage in osteoblasts was cleared similarly with both enzyme types. However, cartilage showed a little response to any of the enzymes. The tagged enzyme reduced storage in cortical neurons, hippocampus, and glia cells. A highly sensitive method of tandem mass spectrometry on serum indicated that the concentration of serum dermatan sulfate and heparan sulfate in mice treated with the tagged enzyme decreased more than the untagged enzyme. These preclinical studies suggest that this AAA-based targeting system may enhance enzyme-replacement therapy.

Enzyme replacement therapy in a murine model of Morquio A syndrome.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to accumulation of keratan sulfate (KS) and chrondroitin-6-sulfate. The pharmacokinetics and biodistributions were determined for two recombinant human GALNSs produced in CHO cell lines: native GALNS and sulfatase-modifier-factor 1 (SUMF1) modified GALNS. Preclinical studies of enzyme replacement therapy (ERT) by using two GALNS enzymes were performed on MPS IVA mice. The half-lives in blood circulation of two phosphorylated GALNS enzymes were similar (native, 2.4 min; SUMF1, 3.3 min). After intravenous doses of 250 units/g body weight were administered, each enzyme was primarily recovered in liver and spleen, with detectable activity in other tissues including bone and bone marrow. At 4 h post-injection, enzyme activity was retained in the liver, spleen, bone and bone marrow at levels that were 20-850% of enzyme activity in the wild-type mice. After intravenous doses of 250 units/g of native GALNS, and 250, 600 or 1000 units/g of SUMF1-GALNS were administered weekly for 12 weeks, MPS IVA mice showed marked reduction of storage in visceral organs, sinus lining cells in bone marrow, heart valves, ligaments and connective tissues. A dose-dependent clearance of storage material was observed in brain. The blood KS level assayed by tandem mass spectrometry was reduced nearly to normal level. These preclinical studies demonstrate the clearance of tissue and blood KS by administered GALNS, providing the in vivo rationale for the design of ERT trials in MPS IVA.