Putative Biological Mechanisms of Efficiency of Substrate Reduction Therapies for Mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by mutations in genes coding for lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Dysfunction of any of these enzymes results in the accumulation of GAGs, which leads to severe clinical symptoms and significantly shortened life span. Several kinds of therapies have been proposed to treat MPS, including bone marrow or stem cell transplantation, enzyme replacement therapy, and gene therapy. Another option is substrate reduction therapy (SRT), in which synthesis of GAGs is inhibited. Recent studies employing in vitro and animal models suggested that this therapy may be efficient in decreasing levels of GAGs in MPS cells, including those bearing two null alleles of the affected gene. Results of behavioral tests in animals as well as some preliminary clinical observations with pediatric patients corroborated the suggestions about possible efficacy of SRT in MPS treatment, including brain functions. Efficient reduction of GAG levels in MPS cells homozygous for null mutations may be intriguing in the commonly accepted scheme of SRT mode of action. In this paper, we propose an explanation of this phenomenon, based on already known facts. Thus, we suggest that SRT may lead to reduction of GAG levels in MPS cells due to inhibition of efficiency of GAG synthesis combined with (a) any readthrough of the stop codon, (b) dilution of already accumulated GAGs due to cell growth followed by cell divisions, and (c) action of endoglycosidases degrading GAGs, e.g., heparanase, in combination with functional GAG-specific hydrolases.     

Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases

Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal storage diseases are monogenic, clear genotype-phenotype correlations occur only in some cases. In this article, we present an overview on various factors and processes, both general and specific for certain disorders, that can significantly modulate expression of phenotypes in these diseases. On the basis of recent reports describing studies on both animal models and clinical data, we propose a hypothesis that efficiency of production of compounds that cannot be degraded due to enzyme deficiency might be especially important in modulation of phenotypes of patients suffering from lysosomal storage diseases.     

Sterility and antibacterial activity of some antibiotics sterilized by irradiation

Sterility and antibacterial activity of several antibiotics (including some penicillins and their salts, gramicidin and neomycin) subjected to sterilization by irradiation has been studied. The compounds in solid phase have been exposed to gamma irradiation in air atmosphere at room temperature, with a dose of 25 kGy, and afterwards they have been subjected to tests recommended by FP V (volume I, 1990) checking their sterility and activity. The results have shown that the majority of initial compounds have been to a slight degree contaminated by bacteria from the genera Bacillus and Micrococcus, the number of bacteria did not exceed 10(2) CFU, and fungi up to 10 CFU in 1 g of the compound. All compounds subjected to sterilization with a dose of 25 kGy were sterile and preserved the activity required by FP V. The decrease in activity observed for some compounds was always within the limits of FP specification. The results have proved that the penicillins analysed, gramicidin and neomycin can be sterilized by irradiation with a dose of 25 kGy, without any detrimental effect on their properties and antibacterial activity.     

New diagnostic criteria for the neuropsychiatric form of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmunologic illness, which apart from changes in the skin, the locomotor system and the internal organs, attacks also the nervous system. The paper presents 19 neuropsychiatric symptomic syndromes which after conduction of multidisciplinary, international research were accepted by the American College of Rheumatology (ACR) as the criteria of systemic lupus erythematosus (SLE). This broadens the criteria applied since 1982, which were only considering acute symptoms and psychoses as characteristic of the neuropsychiatric form of systemic lupus erythematosus (NPSLE). In the new neurologic criteria concerning the CNS are: epileptic attacks and acute attack disorders, headaches, vascular diseases, demyelinating syndrome, aseptical meningitis, chorea, myelopathy. Psychiatric syndromes which make up the new criteria are: acute amentive state, anxiety disorders, cognitive function impairment, affective disorders, psychoses. The criteria connected to the CNS are: cranial nerve damage, mononeuropathy, damage of nerve plexus, polyneuropathy, vegetative neuropathy, myasthenia and acute inflammatory demyelinating polyneuropathy. Clinical symptoms of these syndromes were set and laboratory and visualising tests were developed, which are useful in their diagnosis. The intention of the ACR in setting new, significantly broader criteria of NPSLE, was to stress the diversity of symptoms and for a practical aspect to allow the diagnosis of NPSLE in patients having this disease, in whom the symptoms connected with the nervous system may dominate in the clinical picture, or may be before the dermatological, locomotor or internal organ symptoms.     

Lipoamino acids as major components of absorption promoters in drug delivery

Many biologically active compounds are unsuitable for development as drugs due to their poor bioavailability. For hydrophilic compounds, modifications to increase lipophilicity can increase passive diffusion or increase uptake into the lymphatic system. Alternatively, improved bioavailability of hydrophilic drug candidates may be achieved by formulation with absorption promoters such as surfactants, penetration enhancers, or ion pairing agents. This approach to enhancing bioavailability also has the potential to widen the range of compound categories that can be used as chemical probes to study biological systems in cells and in vivo where membrane permeability would otherwise be a significant limitation. Lipidic amino acids, which combine the structural properties of lipids with those of α-amino acids, represent a relatively unexplored class of agents that can improve drug adsorption. This review discusses the potential of absorption promoters possessing lipoamino acids for improving drug bioavailability.     

In vitro and In vivo evaluation of positively charged liposaccharide derivatives as oral absorption enhancers for the delivery of anionic drugs

Oral delivery of hydrophilic, ionisable drugs remains a major challenge in drug development and a number of active pharmaceuticals fail to reach the market of oral drugs because of a lack of absorption and/or stability issues. One possible approach to improving the bioavailability of such drug candidates is to increase their lipophilicity, which is a key parameter in the permeation across cell membranes. However, modifying the chemical structure by adding lipid residues often results in changes in activity. With ionised molecules, ion‐pairing can be considered to associate charged lipid moieties with the parent drug without altering its structure and therefore activity. This study presents the results of in vitro and in vivo evaluation of a series of synthetic, positively charged liposaccharide derivatives combined with an anionic model drug, piperacillin. The antimicrobial activity, plasma stability, Caco‐2 cell permeability and oral absorption of the conjugates were assessed. Increases in apparent permeability were observed in vitro for three of the tested formulations, while retaining the antibacterial activity of the drug. However, the in vivo intestinal absorption of piperacillin formulated with the liposaccharide derivatives was found unchanged, possibly due to molecular dissociation, early degradation or structural differences between the intestinal epithelium and cultured monolayers. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2333–2342, 2010     

Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses

Sanfilippo disease (mucopolysaccharidosis type III, MPS III) is a severe metabolic disorder caused by accumulation of heparan sulfate (HS), one of glycosaminoglycans (GAGs), due to a genetic defect resulting in a deficiency of GAG hydrolysis. This disorder is characterized as the most severe neurological form of MPS, revealing rapid deterioration of brain functions. Among therapeutic approaches for MPS III, one of the most promising appears to be the substrate reduction therapy (SRT). Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is an isoflavone that has been used in SRT for MPS III. In this report, we tested effects of other flavonoids (apigenin, daidzein, kaempferol and naringenin) on GAG synthesis. Their cytotoxicity and anti-proliferation features were also tested. We found that daidzein and kaempferol inhibited GAG synthesis significantly. Moreover, these compounds were able to reduce lysosomal storage in MPS IIIA fibroblasts. Interestingly, although genistein is believed to inhibit GAG synthesis by blocking the tyrosine kinase activity of the epidermal growth factor receptor, we found that effects of other flavonoids were not due to this mechanism. In fact, combinations of various flavonoids resulted in significantly more effective inhibition of GAG synthesis than the use of any of these compounds alone. These results, together with results published recently by others, suggest that combination of flavonoids can be considered as a method for improvement of efficiency of SRT for MPS III.     

Levels of biological markers of nitric oxide in serum of patients with squamous cell carcinoma of the oral cavity

The aim of the study was a determination of the levels of nitric oxide（NO）and its biological markers such as malonyldialdehyde（MDA）and nitrotyrosine in the serum of patients with squamous cell carcinoma（SCC）of the oral cavity and identification of the relationships between NO and those markers.These studies were performed on patients with SCC of the oral cavity before and after treatment.Griess reaction was used for the estimation of the total concentration of NO in serum.The nitrotyrosine level in serum was assessed with an enzyme-linked immunosorbent assay（ELISA）kit,and MDA level using a spectrophotometric assay.Higher concentrations of NO in blood serum were determined in patients with stage IV of the disease before treatment in comparison to the control group and patients with stages II and III of the disease.Moreover,higher concentrations of MDA and nitrotyrosine were determined in the serum of patients in all stages of the disease in comparison to healthy people.After treatment,lower concentrations of NO in the serum of patients with stage IV of the disease were observed in comparison to the amounts obtained prior to treatment.In addition,lower levels of nitrotyrosine in the serum of patients with all stages of the disease were recorded,whereas higher concentrations of MDA were determined in these patients in comparison to results obtained before treatment.The compounds formed with the contribution of NO,such as MDA and nitrotyrosine,may lead to cancer progression in patients with SCC of the oral cavity,and contribute to formation of resistance to therapy in these patients as well.Moreover,the lack of a relationship between concentrations of NO and MDA,and between NO and nitrotyrosine in serum suggests that the process of lipid peroxidation and nitration in patients with SCC does not just depend on NO.     

iNOS expression and NO production by neutrophils in cancer patients

INTRODUCTION: The tumor-polymorphonuclear neutrophil (PMN) relationship can be altered by the release of toxic molecules, such as nitric oxide (NO). The aim of the present study was to examine the expression of the inducible synthase of NO (iNOS)and NO production by human neutrophils of patients with oral cavity cancer. For comparison we performed similar examinations in autologous peripheral blood mononuclear cells (PBMCs). MATERIAL/METHODS: PMNs and PBMCs were isolated from the whole blood of 27 patients with squamous cell carcinoma of the oral cavity. iNOS protein expression in these cells was detected by Western blot. Total nitrite as an indicator of NO concentrations in the culture supernatants and the serum of patients was measured using a colorimetric assay. RESULTS: The PMNs of oral cavity cancer patients showed a significantly lower intensity of iNOS expression than those of healthy controls. The PBMCs of patients showed a more intensive expression of iNOS than the PMNs, but a lower intensity than the PBMCs of the controls. The expression of iNOS in rhIL-6 and rhIL-15-stimulated PMNs and PBMCs of patients increased in comparison with unstimulated cells. We observed lower productions of NO by PMNs and PBMCs of patients than those of the control group. CONCLUSIONS: The results revealed that altered iNOS expression and NO production are more characteristic of PMNs than of PBMCs of patients with oral cavity cancer. Additionally, this study provided new information about IL-6 and IL-15 activity in a tumor-bearing host.     

The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase,considered as a risk factor for stroke due to increased homocysteine level,can be protected and reactivated by heat shock proteins

The A222 V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222 V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117 V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117 V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222 V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients.