Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.     

Intrathecal enzyme replacement therapy: successful treatment of brain disease via the cerebrospinal fluid

Treatment of brain disease with recombinant proteins is difficult due to the blood-brain barrier. As an alternative to direct injections into the brain, we studied whether application of high concentrations of therapeutic enzymes via intrathecal (IT) injections could successfully drive uptake across the ependyma to treat brain disease. We studied IT enzyme replacement therapy with recombinant human iduronidase (rhIDU) in canine mucopolysaccharidosis I (MPS I, Hurler syndrome), a lysosomal storage disorder with brain and meningeal involvement. Monthly or quarterly IT treatment regimens with rhIDU achieved supranormal iduronidase enzyme levels in the brain, spinal cord, and spinal meninges. All regimens normalized total brain glycosaminoglycan (GAG) storage and reduced spinal meningeal GAG storage by 58-70%. The improvement in GAG storage levels persisted three months after the final IT dose. The successful use of enzyme therapy via the CSF represents a potentially useful approach for lysosomal storage disorders.     

Metabolic activity of osteoarthritic knees correlates with BMI

Osteoarthritis of the knee has consistently been linked to obesity, defined as a body mass index (BMI) > 30 kg/m². It has been hypothesized that obesity may lead to osteoarthritis through increased joint pressure, accumulated microtrauma, and disruption of normal chondrocyte metabolism. These changes in chondrocyte metabolism have not been thoroughly investigated, and it is the purpose of this study to identify a relationship between BMI and altered chondrocyte metabolism in osteoarthritic tissue. Articular cartilage was harvested from the femoral condyles of patients after total knee arthroplasty, and analyzed in explant and alginate models. Glycosaminoglycan (GAG) content was measured using a dimethylmethylene blue assay and normalized to DNA content using a PicoGreen® assay. Studies have reported GAGs to be a reliable measurement of chondrocyte metabolism and osteoarthritis progression. Our results show a significant linear relationship of increasing BMI and increasing GAG content in both alginate and explant models (p < 0.001 and p = 0.001). Obese (BMI ≥ 30 kg/m²) and non-obese (BMI < 30 kg/m²) comparisons also demonstrated significant differences with higher GAG/DNA content in obese individuals compared to non-obese (p = 0.001 and p = 0.015). The study results reveal significant relationships between GAG content and BMI in this population of osteoarthritic patients. The significant difference in GAG content between the obese and non-obese patients supports the connection between osteoarthritis and obesity previously reported. Higher patient BMI (> 30 kg/m²) may be similar to dynamic compression injuries which cause increased GAG synthesis in response to cartilage damage.     

Biodistribution and pharmacodynamics of recombinant human alpha-L-iduronidase (rhIDU) in mucopolysaccharidosis type I-affected cats following multiple intrathecal administrations

The storage disorder mucopolysaccharidosis type I (MPS I) is caused by a deficiency in lysosomal α-L-iduronidase activity. The inability to degrade glycosaminoglycans (GAG) results in lysosomal accumulation and widespread tissue lesions. Many symptoms of MPS I are amenable to treatment with recombinant human α-L-iduronidase (rhIDU), however, peripherally administered rhIDU does not cross the blood-brain barrier and has no beneficial effects in the central nervous system (CNS). A feline model of MPS I was used to evaluate the CNS effects of rhIDU following repeated intrathecal (IT) administration. Twelve animals were randomized into four groups based on the time of euthanasia and tissue evaluation following three repeat IT administrations of 0.1 mg/kg rhIDU or placebo on Study Days 1, 4 or 5, and 9. Two days after the final IT injection, the mean tissue α-L-iduronidase (IDU) activity in the brains of the two treated animals were approximately 3-times higher (50.1 and 54.9 U/mg protein) than the activity found in normal cat brains (mean of 18.3 U/mg), and remained higher than untreated MPSI brain at 1 month (2.4 and 4.1 U/mg protein) before returning to near-baseline levels after 2 months. This activity corresponded with decreased brain GAG concentrations after 2 days (1.4 and 2.0 μg/mg) and 1 month (0.9 and 1.1 μg/mg) which approached levels observed in normal animals (0.7 μg/mg). Attenuation of GAG, gangliosides GM2 and GM3, and cholesterol reaccumulation was identified at both two days and one month following final IT injection. No adverse effects attributable to IT rhIDU administration were observed. IT rhIDU may be an effective means for providing enzyme replacement therapy for the central manifestations of MPS I.     

Glycosaminoglycan storage in neuroanatomical regions of mucopolysaccharidosis I dogs following intrathecal recombinant human iduronidase

Intrathecal (IT) recombinant human α-l-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in mucopolysaccharidosis I (MPS I) dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean: 2.36 ± 0.54 μg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following IT treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared with untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.     

Maroteaux–Lamy syndrome (mucopolysaccharidosis type VI): A single dose of galsulfase further reduces urine glycosaminoglycans after hematopoietic stem cell transplantation

BackgroundMaroteaux–Lamy syndrome, or mucopolysaccharidosis (MPS) type VI, is the autosomal recessive lysosomal disorder resulting from deficient N-acetylgalactosamine 4-sulfatase (ARSB) and the consequent accumulation of glycosaminoglycan (GAG). Patients fully engrafted after hematopoietic stem cell transplantation (HSCT) demonstrate several indicators of metabolic correction such as reduction in liver size, absence of hepatic ultrastructural pathology, and patients do not develop cervical cord compression. Engrafted patients demonstrate reduction in urine GAG achieving near-normal levels.HypothesisWe presumed that HSCT engraftment from a normal individual would provide sufficient systemic enzyme to accomplish maximal metabolic correction, and that no additional benefit would accrue from additional therapy such as with intravenous recombinant human ARSB protein, galsulfase.Materials and methodsA 22-year-old male had received an allogeneic bone marrow transplant from an HLA-identical sibling donor, and remained fully engrafted after 20 years. In response to his request regarding the potential benefit of enzyme replacement therapy, we administered a single, standard dose of galsulfase while monitoring urine GAG daily, before and after the treatment.ResultsUrine GAG declined from slightly high pre-treatment levels (7.63 mg GAG/mmol creatinine; range 7.0–8.5, N = 3) progressively declining below the age-specific normal range (< 6.5) over 10 days to the lowest level of 4.4, with a mean post-treatment level of 5.60 (N = 10).ConclusionsSomewhat surprisingly, the biomarker urine GAG was significantly reduced after a single treatment of intravenous galsulfase thus suggesting that supplemental enzyme replacement therapy might improve the clinical outcome for donor-engrafted patients with MPS VI.     

l-Ascorbic acid partially protects two cycles of cisplatin chemotherapy-induced testis damage and oligo-astheno-teratospermia in a mouse model

Cisplatin (cis-diaminedichloroplatinum-II) is a widely used antineoplastic agent in the treatment of a variety of cancers. The aim of the present study was to investigate the testicular toxicity of cisplatin in mice at human therapeutic dose-levels, and to investigate any protective effects of concomitantly administered l-ascorbic acid (i.p.; 10 mg/kg). Adult male BALB/C mice (13–15-week-old) were treated (i.p.) with two cycles of 5 days each of cisplatin with 17 days of recovery period between cycles, as follows: Group I (G-I) – water (N=10); Group II (G-II) – l-ascorbic acid (N=6); Group III (G-III) – 1 mg/kg (N=6); Group IV (G-IV) – 1 mg/kg+l-ascorbic acid (N=6); Group V (G-V) – 2.5 mg/kg (N=6); and Group VI (G-VI) – 2.5 mg/kg+l-ascorbic acid (N=8). All animals were sacrificed on third day after the last treatment. The testis weight was decreased in a dose-dependent pattern (G-III – 44% and G-V – 54% against G-I), but l-ascorbic acid (10 mg/kg) recovered the lost weight in G-VI up to 32% against G-V (p<0.05). Seminiferous tubular pathology was indicated by vacuoles, epithelial gaps, epithelial sloughing, delayed spermiation, malorientation of spermatids, germ cell degeneration, phagocytosis of spermatids, multinucleated germ cell formation and atrophy. Structurally abnormal tubules (G-III – 33%; G-V – 100%) were induced, and protective effects were seen in G-IV (77%) and G-VI (25%; p<0.05). The tubular diameter was decreased in G-III–VI, but recovery was seen only in G-IV. The epithelial height was decreased in G-III, G-V and G-VI and the recovery was seen only in G-VI. The sperm count was decreased up to 53% and 71% against control in G-III and G-V, respectively, and recovery up to 47% and 61% was observed in G-IV and G-VI, respectively. The sperm motility was decreased up to 56% and 63% against control in G-III and G-V, respectively, and recovery was only marginal in G-IV and G-VI (p>0.05). Total sperm abnormalities were increased in G-III–V (274%, 156% and 232%, respectively, p<0.05) and l-ascorbic acid protected the effect in G-VI up to 156% (p<0.05). In conclusion, at human therapeutic dose-levels, cisplatin induces testicular damage and spermato-toxicity. l-Ascorbic acid only partially nullifies the gonadotoxic effects of cisplatin.     

Assessment of hearing loss by pure-tone audiometry in patients with mucopolysaccharidoses

BackgroundPatients with mucopolysaccharidoses (MPS) often have hearing loss. However, the characterization of hearing loss by pure-tone audiometry (PTA) in this rare disease population and its relationship to age and treatment is limited.MethodsPTA was performed in 39 patients with MPS (29 males and 10 females; 3 with MPS I, 21 with MPS II, 9 with MPS IVA, and 6 with MPS VI; median age, 11.9 years; age range, 4.4–34.2 years). The degree of hearing loss was classified by the age-independent World Health Organization (WHO) clinical guidelines.ResultsHearing loss by PTA was present in 85% (33/39) of patients and was categorized as mild (26–40 dB) in 18%, moderate (41–60 dB) in 36%, severe (61–80 dB) in 23%, and profound (≥ 81 dB) in 5%. Among the patients with hearing loss, 33% were classified as mixed type (conductive and sensorineural), 30% as pure conductive type, 27% as pure sensorineural type, and 9% were undefined. The means of the right and left ear hearing thresholds at 2000 and 4000 Hz by air conduction (AC) and at 500, 1000, 2000, and 4000 Hz by bone conduction (BC) were all positively correlated with age (p < 0.05). In the 6 patients with MPS II or VI who underwent follow-up PTA after ventilation tube insertion and enzyme replacement therapy for 1.9 to 8.5 years, all showed improvements in AC and BC of the better ear, as well as in the air-bone gap.ConclusionsHearing impairment is common in MPS. Early otolaryngological evaluation and intervention are recommended. These findings and the follow-up data can be used to develop quality of care strategies for patients with MPS.     

A dose-optimization trial of laronidase (Aldurazyme®) in patients with mucopolysaccharidosis I

Recombinant human α-l-iduronidase (Aldurazyme^®, laronidase) is approved as an enzyme replacement therapy to treat the lysosomal storage disorder, mucopolysaccharidosis type I (MPS I) at a dose of 0.58 mg/kg by once-weekly intravenous infusion. To assess whether alternate dosing regimens might provide a better reduction in lysosomal storage, a 26-week, randomized, open-label, multinational dose-optimization trial was conducted. The pharmacodynamic effect and safety of the approved laronidase dose was compared to three alternative regimens (1.2 mg/kg every 2 weeks; 1.2 mg/kg every week; 1.8 mg/kg every 2 weeks) among 33 MPS I patients. The four treatment regimens showed no significant differences in the reduction of urinary glycosaminoglycan excretion or liver volume. Laronidase had an acceptable safety profile in all dose regimen groups. Infusion-associated reactions were the most common drug-related adverse events across dose regimens (by patient incidence), and included pyrexia (21%), vomiting (15%), rash (15%), and urticaria (12%). Patients in the approved dose group had the lowest incidence of drug-related adverse events (38% vs. 63–75%) and infusion-associated reactions (25% vs. 25–63%). There was one death: a patient with acute bronchitis died of respiratory failure 6 h after completing the first laronidase infusion. The approved 0.58 mg/kg/week laronidase dose regimen provided near-maximal reductions in glycosaminoglycan storage and the best benefit-to-risk ratio. The 1.2 mg/kg every 2 weeks regimen may be an acceptable alternative for patients with difficulty receiving weekly infusions, but the long-term effects of this regimen are unknown.     

Effects of l-ascorbic acid on two cycles of cisplatin-induced DNA double-strand breaks and phosphorylation of p53 in the liver

Cisplatin, a commonly used anticancer drug, was studied to investigate its effects on structure, DNA damage and p53 along with the possible protective effects of l-ascorbic acid in the liver. Adult male BALB/c mice were treated with 0, 10 mg/kg l-ascorbic acid and two cycles of cisplatin 1 mg/kg/2.5 mg/kg with or without l-ascorbic acid (17 days recovery period between the cycles) and the livers were collected at 72 h after the last exposure. Structural damage was analyzed in Masson's trichrome and Hortega's silver stained liver tissues. The DNA double-strand breaks with duplex 3′ overhangs and 5′ P-blunt ends were labeled by in situ oligo ligation by using hairpin oligonucleotide probes. The expression of p53 and phosphorylated p53 (p-p53) was detected by immunohistochemistry. Structural changes such as vacuolization of hepatocytes, pyknosis, infiltration of leukocytes and pericentral fibrosis were observed without any protection from l-ascorbic acid. The reticular fibrous framework was affected and the incidence of Kupffer cells was decreased. Cisplatin induced the DNA double-strand breaks (p < 0.001); however, the latter appeared in a p53-independent, but p-p53-dependent manner. l-ascorbic acid showed significant protective effect on cisplatin-induced DNA damage (p < 0.001). Cisplatin also enhanced p53 phosphorylation in a dose-dependent manner and l-ascorbic acid reduced this biochemical change only in 1 mg/kg group. In conclusion, cisplatin-induced structural changes are not, but the DNA damage and phosphorylation of p53 are, significantly, but not completely, alleviated by l-ascorbic acid.     

Protective effects of nigella sativa against gentamicin-induced nephrotoxicity in rats

The aim of this study was focused on investigating the possible protective effect of NS against GS-induced nephrotoxicity. Twenty four Wistar-albino rats were divided into four equal groups as follows: control group, GS group (100 mg/kg intraperitoneal – i.p.), NSL+GS group (0.2 ml/kg+100 mg/kg i.p.) and NSH+GS group (0.4 ml/kg+100 mg/kg i.p.). Plasma creatinine and urea levels significantly increased as a result of nephrotoxicity in the GS group. Also, creatinine and urea levels significantly decreased in NSL+GS and NSH+GS groups. In the GS group, plasma MDA and NO levels increased significantly (p<0.05) and erythrocyte SOD and GSH-Px activities decreased significantly (p<0.05) when compared with control group. NS administration with GS injection resulted in significantly decreased MDA and NO generation and increased SOD and GSH-Px activities when compared with GS group. Proximal tubular necrosis, vacuolation, desquamation and degeneration in epithelial cells of the proximal tubules, hyaline casts in tubular lumen, mononuclear cell infiltration, glomerular and basement membrane alterations were histopathologically detected in the kidneys of the GS group. Co-treatments with NS (low and high dose) considerably decreased the renal damage when compared with the GS group. In conclusion, NS acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GS both in the biochemical and histopathological parameters.     

Effects of N-methyl-d-aspartate (dizocilpine) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat

The present study was undertaken to examine the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3–10 mg/kg, i.p.), a competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34–53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1–3 mg/kg, i.p.), a non-competitive N-methyl-d-aspartate receptor antagonist, also significantly decreased the urine release (62–82%) and increased residual volume (180–230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%.These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.     

Kenaf seed supercritical fluid extract reduces aberrant crypt foci formation in azoxymethane-induced rats

Kenaf (Hibiscus cannabinus) a plant of the family Malvaceae, is a valuable fiber plant native to India and Africa. Kenaf seeds contain alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E and other antioxidants with chemopreventive properties. In the present study we examined the hypothesis that kenaf seed ‘supercritical fluid extract’ (SFE) extract could suppress the early colon carcinogenesis in vivo by virtue of its bioactive compounds. To accomplish this goal, 60 male rats were randomly assigned to 5 groups which were (1) negative control group [not induced with azoxymethane (AOM)]; (2) positive control group (induced with AOM but received no treatment); (3) group treated with 500 mg/kg kenaf seed SFE extract; (4) group treated with 1000 mg/kg kenaf seed SFE extract; (5) group treated with 1500 mg/kg kenaf seed SFE extract. At 7 weeks of age, all rats except the negative control group received 15 mg/kg of AOM injection subcutaneously once a week for 2 weeks. Rats were euthanized at 13 weeks of the experiment. Number of ACF (mean ± SD) ranged from 84.4 ± 4.43 to 179.5 ± 12.78 in group 2, 3, 4, 5. ACF reductions compared with the untreated group were 45.3, 51.4 and 53.1% in rats fed with 500, 1000 and 1500 mg/kg body weight, respectively. There were no significant differences in weight gain among groups. Our finding indicates that kenaf seed SFE extract reduced AOM-induced ACF in Sprague–Dawley male rats.     

Immunohistochemical and histological evaluations of cyclophosphamide-induced acute cardiotoxicity in wistar rats: The role of turmeric extract (curcuma)

Chemotherapy-induced cardiac derangement is a major concern in health sector. Cyclophosphamide as a chemotherapeutic agent induces acute cardiotoxicity through its toxic metabolite, acrolein. This study evaluated the effect of ethanol extract of turmeric on cyclophosphamide-induced acute cardiotoxicity in Wistar rats. Thirty-five healthy Wistar rats, weighing 200–250 g were randomly assigned into 7 groups (Groups A, B, C, D, E, F and G) N = 5. Group A was the control, group B was negative control, and group C was administered 200 mg/kg of turmeric extract (orally) only. While groups B, D, E, F and G were all administered 100 mg/kg cyclophosphamide (i.p) for 10 days. Groups D and E were administered 100 mg/kg and 200 mg/kg of turmeric extract (orally) respectively for 72 hours before cyclophosphamide administration. Groups F and G were concomitantly administered 100 mg/kg cyclophosphamide (i.p) with doses of 100 mg/kg and 200 mg/kg of turmeric extract (orally) respectively. The rats were sacrificed under ketamine anesthesia (30 mg/kg i.m). The left ventricle of the heart was excised. One-way ANOVA was used to analyze data. Results revealed that there was statistically significant (P < 0.05) difference in body weight change, CK–MB, and LDH across all experimental groups; which were significantly lower in cyclophosphamide group. Histology and Immunohistochemistry revealed that there were morphological alterations in the myocardium of the left ventricle in group B while turmeric extract ameliorated cyclophosphamide-induced damage in the myocardium in other experimental groups. In conclusion, cyclophosphamide-induced myocardial alterations were significantly ameliorated through administration of ethanol extract of turmeric.     

Effect of quercetin against dichlorvos induced nephrotoxicity in rats

This study was carried out to determine the effect of quercetin against renal injury induced by dichlorvos (DDVP) in rats. Rats were randomly assigned to control, DDVP-treated (7.2 mg/kg bw), three different doses of quercetin-treated (2 mg/kg bw, 10 mg/kg bw, 50 mg/kg bw) and different doses of quercetin plus DDVP-treated groups. DDVP was administered daily to rats through their drinking water, and quercetin was administered by intragastric gavage for 90 days. By the end of the 90th day in the DDVP-treated group, the following indices significantly increased compared with the control (P < 0.01): activities of catalase, glutathione peroxidase and superoxide dismutase; level of malondialdehyde in kidney tissues; serum levels of creatinine and urea nitrogen; and level of β2-microglobulin, level of retinol-conjugated protein, and activity of N-acetyl-β-d-glucosaminidase in urine; by contrast, urine uric acid levels significantly decreased. However, in the quercetin (50 mg/kg bw) plus DDVP group, the aforementioned indices were significantly decreased compared with the DDVP-treated group (P < 0.05), except the urine uric acid levels were significantly increased (P < 0.05). Thus, rat exposure to DDVP caused renal injury, including renal tubular, glomerular filtration, and oxidative stress. These toxic effects were also regulated by high-dose quercetin. Histopathological examination revealed that exposure to DDVP induced extensive cell vacuolar denaturation, but milder histopathological alterations in the kidney tissues of rats co-treated with DDVP and quercetin (50 mg/kg bw) were observed. These results indicated that quercetin at 50 mg/kg bw can partly prevent the kidney injury induced by DDVP.     

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model

The cyclobutylglycine (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1α and 5α, with IC₅₀ values of 1.0±0.4 μM and 1.6±1.4 μM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent K_d values were estimated to be 0.3 μM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 μM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root–ventral root potential elicited at 8× threshold was depressed by LY393053 with IC₅₀ values of 9.0±0.7 μM and 12.7±1.7 μM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1–10 mg/kg intraperitoneally. An ED₅₀ value of 6.0 mg/kg was obtained in this test.By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.     

Antidiabetic,antihyperlipidemic and antioxidant effects of the flavonoid rich fraction of Pilea microphylla (L.) in high fat diet/streptozotocin-induced diabetes in mice

The present study describes the antidiabetic effect of the flavonoid rich fraction of Pilea microphylla (PM1). HPLC characterization of PM1 revealed the presence of polyphenols viz., chlorogenic acid, rutin, luteolin-7-O-glucoside, isorhoifolin, apigenin-7-O-glucoside, and quercetin. PM1 inhibited dipeptidyl peptidase IV (DPP-IV) in vitro with an IC₅₀ of 520.4 ± 15.4 μg/ml. PM1, at doses of 300, 600 and 900 mg/kg i.p., also produced dose-dependent mean percent reductions of 9.9, 30.6 and 41.0 in glucose excursion (AUC_0–120 min) respectively in lean mice. However, even the highest dose of PM1 did not alter normoglycemic condition. PM1 at dose of 100 mg/kg/day, i.p. for 28 days produced significant (p < 0.05) reduction in body weight, plasma glucose (PG), triglycerides (TG) and total cholesterol (TC) content in high-fat streptozotocin-induced diabetic mice. PM1 also improved oral glucose tolerance significantly (p < 0.05) with mean percentage reduction of 48.0% in glucose excursion (AUC_0–120 min) and significantly (p < 0.05) enhanced the endogenous antioxidant status in mice liver compared to diabetic control. PM1 preserved islet architecture and prevented hypertrophy of hepatocytes as evident from the histopathology of pancreas and liver. PM1 did not show any detectable hematological toxicity at therapeutic doses. In conclusion, PM1 exhibits antidiabetic effect possibly by inhibiting DPP-IV and improving antioxidant levels in high fat diet/streptozotocin (HFD/STZ) diabetic mice.     

Dose-dependent hepatoprotective effect of emodin against acetaminophen-induced acute damage in rats

Protective effect of emodin (1,3,8-trihydroxy-6-methyl anthraquinone), an active compound of Ventilago madraspatana Gaertn., was evaluated against acetaminophen-induced biochemical and histological alterations in rats. Acetaminophen (2 g/kg, po) administration caused significant elevation in the release of serum transaminases, alkaline phosphatase, lactate dehydrogenase, serum bilirubin and serum protein with concomitant decrease in hemoglobin and blood sugar after 24 h of its administration. Toxicant exposure intensified the lipid peroxidation and altered glutathione status, activities of adenosine triphosphatase, acid phosphatase, alkaline phosphatase as well as major cellular constituents i.e., protein, glycogen and total cholesterol in liver and kidney. Treatment of emodin (20, 30 and 40 mg/kg, po) significantly lessened the toxicity by protecting acetaminophen-induced alterations in various blood and tissue biochemical variables after 24 h of its administration. Acetaminophen administration initiated histological damage in liver. Some degree of protection was seen after emodin therapy in a dose-dependent manner. Emodin at doses of 30 and 40 mg/kg effectively reversed toxic events induced by acetaminophen as same as silymarin (50 mg/kg, po). Thus, the study concluded that emodin at a dose of 30 mg/kg (po) possesses optimum hepatoprotective ability against acetaminophen-induced toxicity.     

Effects of compound X,a novel potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase,on the adrenal gland of rats

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150 mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150 mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300 mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10 mg/kg, and eosinophilic changes in the ZF at 150 mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30 mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300 mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.     

Neuroprotective effect of lamotrigine and MK-801 on rat brain lesions induced by 3-nitropropionic acid: evaluation by magnetic resonance imaging and in vivo proton magnetic resonance spectroscopy

Magnetic resonance imaging and in vivo proton magnetic resonance spectroscopy were used to evaluate the therapeutic effect of lamotrigine and MK-801 on rat brain lesions induced by 3-nitropropionic acid. Systemic administration of 3-nitropropionic acid (15 mg/kg per day) to two-month-old Sprague–Dawley rats (n=10 for each group) for five consecutive days induced selective striatal and hippocampal lesions and specific behavioral change. Pretreatment with lamotrigine (10 mg/kg or 20 mg/kg per day) or MK-801 (2 mg/kg per day) attenuated the lesions and behavioral change. There were no significant differences in T2 values of the striatum and hippocampus among rats pretreated with MK-801, lamotrigine (20 mg/kg) and sham controls. Significant elevations of succinate/creatine and lactate/creatine ratios and decreases of N-acetylaspartate/creatine and choline/creatine ratios were observed after 3-nitropropionic acid injections (P<0.001). The changes were nearly prevented after pretreatment with lamotrigine (20 mg/kg). However, the N-acetylaspartate/creatine in rats pretreated with lamotrigine (10 mg/kg) (P<0.01) and MK-801 (P<0.05) still showed significant reduction as compared with sham controls.Thus we conclude that both lamotrigine and MK-801 are effective in attenuation of brain lesions induced by 3-nitropropionic acid. A higher dose of lamotrigine provides a better neuroprotective effect than MK-801. With a better therapeutic effect and fewer side effects, lamotrigine is more promising for potential clinical application.