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Fusidic acid (FA) had excellent antimicrobial effects due to its unique mechanism of action. Since 1962, FA has been widely used in the systemic and topical treatment of staphylococcal infections and exhibits a well-characterized potency against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant coagulase-negative Staphylococci. In view of the spectrum of activity, no cross-resistance with other clinically used antibiotics, and potential penetration into brain tissue, FA was used to treat possible gra-positive bacteria in 3 patients with intracranial infections in the present report. FA and its active metabolite (3-keto FA) were measured in plasma and cerebrospinal fluid (CSF) to assess the treatment of FA, and the results indicated that 1,500 mg per day of FA was sufficient to achieve therapeutic concentrations in both plasma and CSF in intracranial infection patients, while the dosage did not experience unexpected regimen-related toxicity.

Background Critically ill patients show several pathophysiological alterations that can complicate antibiotic dosing. Hence, there is a strong rationale to individualize anti-infective dosing in these patients by using therapeutic drug monitoring (TDM). The current study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound plasma concentrations of 3 commonly used antibiotics (meropenem, imipenem/cilastatin, and cefoperazone/sulbactam) in the treatment of infections in critically ill patients in China, which could be suitable for routine TDM in hospital laboratories. Methods The unbound drug was separated from the bound drug by ultrafiltration. Simple protein precipitation was used for sample preparation. Meropenem, imipenem/cilastatin, cefoperazone/sulbactam, and their corresponding internal standards were then resolved using the Waters CORTECS C18 column. All the compounds were detected using electrospray ionization in the positive/negative ion-switching mode. Results The calibration curves were linear for all compounds, with correlation coefficients (R) above 0.99 for total concentrations in human plasma and unbound concentrations in the plasma ultrafiltrate. For both total and unbound drugs, the relative errors and intra-assay/interassay relative standard deviations were below 15%. The limit of quantification was 0.05 mcg/mL for both total plasma concentrations and plasma ultrafiltrate concentrations of all compounds. Conclusions The method was simple, rapid, and reliable and is currently being used to provide a TDM service to enhance the efficacious use of the 3 antibiotics.

Isoniazid (INH)-induced liver injury may be associated with inhibition of the liver farnesoid X receptor (FXR). However, the relationship between FXR and INH-induced liver injury remained unclear. The present study was performed to clarify the role of inhibition of FXR in the pathogenesis of INH-induced liver injury and to further identify potential inhibitors of FXR from INH and its metabolites. HepaRG cells were treated with INH (10 mM) plus mixed bile acids (BA) and rats were treated with INH (60–600 mg/kg p.o.) or INH plus obeticholic acid (OCA, 10 mg/kg), a potent FXR agonist, for seven days. INH can cause BA-dependent toxicity and apoptosis with elevated intracellular bile acids in vitro; indeed, in these studies, liver bile acids and mRNA levels for Cyp7a1, an FXR target gene were increased, while mRNA levels for FXR and Shp were significantly decreased, and these changes could be prevented by co-treatment with the FXR agonist OCA. In silico molecular docking studies showed that INH, acetyl isoniazid, isonicotinic acid and PIH may be potential FXR inhibitors, and a TR-FRET FXR-coactivator assay confirmed that PIH is a strong antagonist of FXR (IC50 = 52 nM). To further determine if PIH also inhibits FXR activity in vivo, rats were treated with PIH directly (5 mg/kg). Liver total bile acids were significantly increased while FXR expression was not changed, but Shp mRNA levels were significantly decreased and Cyp7a1 mRNA was significantly increased, consistent with PIH acting as an FXR antagonist. In summary, PIH inhibition of liver FXR function leading to bile acid accumulation in hepatocytes may be an early pathogenesis event in INH-induced liver injury.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 → 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.

Objectives Gardenia Jasminoides Ellis (Zhizi), belonging to Rubiaceae family, has been traditionally used for treatment cholestasis and jaundice for centuries in Asian countries. In the theory of Traditional Chinese Medicines, Zhizi could dispel dampness and heat via the urine to execute its choleretic effects. However, the potential molecular mechanism has been still poorly clarified. Here, we investigated the effect of different dose of Zhizi aqueous extract powder (0.3 g/kg/d and 0.9 g/kg/d) on urinary excretion of bile acids (BAs), and defined the potential mechanism via renal BAs efflux transporters Mrp2 and Mrp4 in normal rats. Methods Male Wistar rats were orally administrated with 0.3 or 0.9 g/kg/d dose of Zhizi aqueous extract powder for 2 weeks, then body weight, serum aminotransferase, total BAs concentrations in liver, bile, serum, kidney and urine, 1 h bile flow, 12-h urinary volume, biliary and urinary excretion amount of total BAs as well as protein expression of major renal BAs efflux transporter Mrp2 and Mrp4, were all evaluated. Results Zhizi especially the high dose of Zhizi aqueous extract powder could reduce hepatic total BAs concentration. Additionally, bile flow and biliary excretion had no significant difference, but the remarkable increasing urinary excretion of BAs and 2 to 3 folds up-regulated renal Mrp2 expression were observed after administrated with Zhizi as compared with the control group. Conclusion The findings indicate that Zhizi reduces hepatic total BAs level by increasing urinary excretion rather than the biliary excretion of BAs, which, in turn ascribed to elevated protein expression of Mrp2 at apical membrane of renal tubular epithelial cells.

Isoniazid (INH) and rifampicin (RFP), two front-line drugs used in tuberculosis therapy, may lead to seriously hepatotoxicity. The current study was carried out to investigate the hepatoprotective effects of hesperidin against INH- and RFP-induced oxidative damage. The liver injury animal model of rats was induced by INH (75 mg/kg) and RFP (150 mg/kg) coadministration for 7 days, and hesperidin, at the dose of 50, 100, and 200 mg/kg, was orally administered to rats 2 h before INH and RFP administration. The biochemical and pathologic examinations were performed after rats were sacrificed. Moreover, the serum and liver glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), GSH peroxidase, and GSSG reductase were determined by test kits, and the expression of multidrug resistance proteins 2 (Mrp2) was determined by Western blotting and immunohistochemistry. The results showed that hesperidin significantly alleviated liver injury as indicated by the decreased levels of ALT, AST, bilirubin, total bile acid, and glutathione peroxidase and the increased levels of the GSH/GSSG ratio and the expression of Mrp2. Moreover, hesperidin could effectively reduce the pathological tissue damage. These results indicate that hesperidin can attenuate INH-and RFP-induced oxidative damage, and the underlying mechanism may have correlation with its effect on the upregulation of Mrp2. (C) 2016 Wiley Periodicals, Inc.

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome.

Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).

A simple, specific and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), -hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC-C-18 column (4.6x250mm, 5 mu m) at a flow rate of 1.0mL/min, and post-column splitting (1:4) was used to give optimal interface flow rates (0.2mL/min) for MS detection; the total run time was 8.5min. Mass spectrometric detection was achieved using a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42-7000ng/mL for MET, 2.05-4200ng/mL for HMT and 1.95-4000ng/mL for DMT. The analytical method was successfully applied to herb-drug interaction study of MET and breviscapine after administration of breviscapine (12.5mg/kg) and MET (40mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright (c) 2015 John Wiley & Sons, Ltd.

Venlafaxine (VLX) could be pumped out of the brain by P-glycoprotein (P-gp). Moreover, the expression of P-gp distributed in blood-brain barrier could be significantly induced by VLX. Thus, P-gp could be considered as the nature barrier for delivering of VLX to the brain. The aim of this study was to investigate whether the efflux function and increased expression of P-gp could be reversed by utilizing solid lipid nanoparticles (SLN). VLX solid lipid nanoparticles (VLX - SLN) were prepared and evaluated. Pharmacokinetics and brain distribution of VLX in different formulations were conducted after oral or intravenous administration. P-gp efflux function to VLX was evaluated by the brain uptake amount of VLX, while P-gp expression was investigated by Western blotting. Results indicated that the entrapment, mean size and zata potential of VLX - SLN was 74.9 ± 3.0 %, 186.3 ± 69.26 nm and -22.8 ± 7.78 mv, respectively. After vein injection of VLX formulations, the brain uptake amount of VLX from VLX - SLN was significantly higher than that of VLX solution, VLX solution with empty SLN (VLX+ empty SLN) and VLX solution with Verapamil (VLX + Ver), respectively. Furthermore, the protein mass of P-gp in VLX - SLN treated group was the lowest among all the investigated groups. These results indicated that SLN could overcome P-gp and achieve brain target by intravenous administration.

Yin-zhi-huang (YZH) injection is an injectable multi-herbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multi-herbal prescription is still challenged by desirable analytical methods, by which a big array of components could be simultaneously detected at low concentrations. Herein, a new liquid chromatography- tandem mass spectrometry (LC-MS/MS) method by using dynamic multiple reaction monitoring (DMRM) mode was developed to determine multiple peaks in traditional Chinese medicine (TCM) preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with the lower limit of quantification (LLOQ) of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and/or binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates would be used to investigate the pharmacodynamics in the further study.

In this review, we have highlighted the adverse drug reaction mediated by transporters from two aspects: (1) competitive interactions between drug and drug/metabolite/endogenous substance mediated by transporters; (2) the expression/function change of transporter due to physiologic factors, disease, and drugs induction. It indicated that transporters exhibited a broad substrate specificity with a degree of overlap, which could change the pharmacokinetics of drugs and cause toxicity due to competition interactions among substrates. In addition, the expression and function of transporters were regulated by physiological conditions, pathological conditions, and drugs induction, which could cause adverse drug reaction and interindividual differences. Furthermore, one substrate was always medicated by several transporters and often subjected to metabolism by CYP enzymes, so we should be more aware of the increased plasma concentration of drugs caused by drug transporters as well as drug metabolizing enzymes synergistically, especially for drugs with narrow therapeutic window. In addition, the weightiness for one transporter to induce drugs plasma/tissue concentration change could be different in different condition. On the whole, transporters were corresponding with systemic/organs exposure of drug/metabolites/endogenous compounds. So understanding the expression and function in drug transporters will result in better strategies for optimal dosage regimen and reduce the risk for drug adverse reaction as well as adverse drug-drug interactions.

Background and purpose Long-term medical consequences of cholecystectomy are believed to be uncommon. It has been reported that bile acids (BAs) changed after cholecystectomy. As important signaling molecules, the alternations of BAs might favour the regulatory effect on enzymes and transporters involved in BAs physiological homeostasis at the transcriptional level, which could lead to pharmacokinetic changes of drugs. Here, we determined the effect of cholecystectomy on BAs, relevant enzymes and transporters and pharmacokinetic parameters of rifampicin, and explored the potential mechanisms at the transcriptional regulatory level via nuclear receptors. Methods Parameters of BAs in different specimens, mRNA and protein expression of enzymes, transporters and nuclear receptors that relate to BAs homeostasis in liver and ileum, and the pharmacokinetic character of rifampicin were measured in sham-operated and cholecystectomized mice. Key results Cholecystectomy associated with considerable decreased BAs pool size that could attribute to increased fecal excretion. Most notably, as the Fxr and Pxr ligands, the alternations of hepatic and ileal individual BAs affected expression of enzymes Cyp3a11 and transporters Ntcp and Bsep in liver and Asbt in ileum significantly following cholecystectomy. Eventually, the rifampicin bioavailability was improved with depressed clearance in mice without gallbladders. Conclusion As natural ligands for Fxr and Pxr, the alternations of individual BAs lead to the regulation of hepatic and ileal relevant enzymes and transporters after cholecystectomy. Especially, the down-regulation of hepatic Cyp3a11 suggests that undesirable pharmacokinetic alternations of drugs especially Cyp3a11 substrates like rifampicin might occur in phase with cholecystectomy.

Organic cation transporter 2 (rOCT2) and multidrug and toxin extrusion protein 1 (rMATE1) are mainly expressed in rat renal proximal tubules and mediate urinary excretion of cationic drugs, such as metformin. Accumulated evidence indicated that renal rOCT2 expression in male rats is much higher than that of female rats. However, it is unclear whether the gender-related differences in rOCT2 expression between male and female rats can affect the urinary excretion of metformin. The aim of this study was to investigate the effect of gender on the pharmacokinetics of metformin and to clarify the effect of gender-related differences on renal rOCT2 expression and its role in urinary excretion of metformin. Renal rOCT2 levels, but not rOCT1 and rMATE1, were significantly lowered in female rats when compared to that of male rats (P 0.01), while the pharmacokinetic parameters, i.e., AUC0→t, t 1/2, CL/F, and cumulative urinary excretion of metformin, did not show any significant differences between female and male rats following oral administration of metformin at l00 mg/kg (P 0.05). However, when metformin was orally administered at the dose of 500 mg/kg, the cumulative urinary excretion and renal tissue-to-plasma concentration ratio of metformin in female rats (26,689 ± 1266 μg and 2.96 ± 0.47 mL/g, respectively) were markedly lowered compared to that of male rats (32,949 ± 1384 μg and 4.20 ± 0.31 mL/g, respectively), and the plasma concentration of metformin in female rats (55.9 ± 4.5 μg/mL) was significantly increased compared to that of male rats (43.5 ± 3.1 μg/mL) at 2 h after oral administration. These results indicated that effect of gender-related differences on renal rOCT2 expression indeed contributes to the decreased urinary excretion of metformin in female rats when metformin was administered at relatively high doses.

Yin-Zhi-Huang (YZH), a prescription of traditional Chinese medicine, is widely used to treat neonatal jaundice or cholestasis.This study investigates the regulatory effect of YZH on the localization and expression of organic anion transporting polypeptides 2 (Oatp2), Na(+)-taurocholate co-transporting polypeptide (Ntcp), multidrug-resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) in estrogen-induced cholestasis rats.Cholestasis model rats were induced via subcutaneous injection of estradiol benzoate (EB, 5 mg/kg/d) for 5 d. Other EB-induced rats were treated with saline (2 ml) or YZH (1.5 g/kg, two times a day) for 7, 14, and 21 d. The biochemical and pathologic examinations were performed. Moreover, the localization and expression of Oatp2, Ntcp, Mrp2, and Bsep were determined by immunohistochemisty and Western blotting, respectively.YZH treatment could significantly decrease the serum total bile acids (TBA) (4.9 ± 0.6-2.8 ± 0.8) and direct bilirubin (DBIL) (2.6 ± 0.7-1.0 ± 0.1) levels, improve the histological disorganization, and, respectively, increase the expression of Oatp2 and Ntcp by 46% and 28% compared with saline-treated (p0.05) rats at 14 d. The expression of Mrp2 increased by 45% was observed in YZH treated compared with saline-treated (p0.05) rats at 7 d. However, there was a little change in the expression of Bsep (p0.05) after YZH treatment for 7, 14, and 21 d.In conclusion, the therapeutic effect of YZH to cholestasis could be attributed to the regulation of Oatp2, Ntcp, Mrp2, and Bsep.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Objective: To investigate whether traditional Chinese herbal formula Yupingfeng (YPF) powder has an anti-inflammatory effect on colonic inflammation, and to explore the mechanism involved. Materials and Methods: YPF powder was orally administrated to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice at the dose of 3, 6, and 12 g/kg/d for 7 consecutive days. Body weight, stool consistency, histopathological score, and myeloperoxidase (MPO) activity were tested to evaluate the effect of YPF powder on colonic inflammation while colonic enterochromaffin (EC) cell density and serotonin 5-hydroxytryptamine (5-HT) content were investigated to identify the effect of YPF powder on colonic 5-HT availability. Results: The results showed that the body weight of colitis mice was markedly decreased by 10, 12, 14, and 17% at 1, 3, 5, and 7 days (P < 0.05), whereas stool consistency score (3.6 vs. 0.4, P < 0.05), histopathological score (3.6 vs. 0.3, P < 0.05), and MPO activity (2.7 vs. 0.1, P < 0.05) in colitis mice were significantly increased compared to that of the normal mice; YPF powder treatment dose-dependently increased the body weight (7–13% increase) and decreased the stool consistency score (0.4–1.4 decrease), histopathological score (0.2–0.7 decrease), and MPO activity (0.1–0.9 decrease) in colitis mice. Colonic EC cell density (70% increase) and 5-HT content (40% increase) were markedly increased in colitis mice (P < 0.05), YPF powder treatment dose-dependently reduced EC cell density (20–50% decrease), and 5-HT content (5–27% decrease) in colitis mice. Conclusion: The findings demonstrate that the anti-inflammatory effect of YPF powder on TNBS - induced colitis may be mediated via reducing EC cell hyperplasia and 5-HT content. The important role of YPF powder in regulating colonic EC cell number and 5-HT content may provide an alternative therapy for colonic inflammation.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder that affects both the central and peripheral nervous system (CNS and PNS). The deficiency of the lysosomal enzyme galactocerebrosidase (GALC) leads to the accumulation of psychosine, resulting in the death of oligodendrocytes and Schwann cells. Bone marrow transplantation (BMT) of the mouse models of GLD can prolong their lives up to one year compared to 40-50 days if untreated. Intracranial and intraventricular injections of adeno-associated virus (AAV) types 1 and 5 containing mouse GALC cDNA into affected mice results in widespread GALC expression in the brain, pathological improvement and modest extension of life span compared to untreated mice. Mice treated with either BMT or viral therapy still demonstrate mild twitching and premature death at least partially due to lack of correction of PNS pathology. In an attempt to improve the biochemical and pathological condition of the peripheral nerves in affected mice, AAV2/1-mGALC viral particles were injected into the gastrocnemius muscles of affected mice. Biochemical analysis of the different tissues from the treated mice demonstrated very high GALC activity in the injected muscle and significantly increased GALC activity in the contralateral muscles. This was confirmed by histochemical staining of GALC activity where a large number of myofibers and the sciatic nerve in the injected side were strongly positive. The sciatic nerve on the contralateral side was also positive for GALC activity. While the sciatic nerve of untreated affected mice was thickened due to edema, initial studies following intramuscular injection showed that the sciatic nerve was much thinner and similar to control mice. Sections from the spinal cord also demonstrated some GALC-positive motoneurons indicating retrograde transfer of AAV2/1-GALC from the intramusclar injections. Transgene expression in the injected muscle was still very strong three months after injection. Combining intramuscular injections with other therapeutic approaches that improve the CNS pathology may have additional advantages in the successful treatment of the animal models of GLD.