Impact of discontinuing 5 years of enzyme replacement treatment in a cohort of 6 adults with hypophosphatasia: A case series.

Asfotase alfa is a human recombinant enzyme replacement therapy for hypophosphatasia. We describe 6 adults who were treated with asfotase alfa for 61-68 months in a clinical trial (NCT01163149), after which asfotase alfa was discontinued for 15-48 months. The patients experienced clinical deterioration and, when treatment was restarted, showed improvement. Patients with hypophosphatasia should be closely monitored if asfotase alfa is stopped as clinical decline is likely. Clinical practice guidelines are needed.

Early diagnosis and successful long-term management of a rare, severe lysosomal acid lipase deficiency/Wolman disease patient: Infancy to age five.

Background: This report describes a unique case of long-term survival of a young girl who was diagnosed with severe, rapidly progressive lysosomal acid lipase deficiency (LAL-D; historically "Wolman disease") at three months of age and began receiving therapeutic interventions at four months of age. This disease involves rapidly progressive multisystemic impairments and limited survival (6-12 months) without treatment. Methods: Case report taking into account clinical aspects and patient management including a semi-structured interview with the main family caregiver. Results: Presentation at two months of age: severe malnutrition and chronic diarrhea; hypoalbuminemia; low iron, vitamin A, and vitamin D levels; high triglyceride levels; profound anemia; thrombocytopenia; adrenal calcifications; and mild hepatosplenomegaly. Enzyme replacement therapy (ERT) with sebelipase alfa, parenteral nutrition, and a low-fat diet began at age four months. The patient has received sebelipase alfa for >5 years with good tolerability and is thriving, with a body mass index of 16.35 kg/m2 (80th percentile) despite a stature delay (height <3rd percentile), and mild developmental delay. Optimal medical management requires that family caregivers and health professionals have the knowledge and skills to provide appropriate care and supports multidisciplinary teams through transfer of knowledge to all stakeholders. Effective coordination of services and activities related to child health and development, including navigation of administrative and financial barriers, is also imperative. Conclusions: Formerly fatal in untreated infants, severe LAL-D, when diagnosed early, can be promptly and effectively treated by combining sebelipase alfa ERT, modified diet, involvement of family caregivers, and multidisciplinary team collaboration.

Clinical Characteristics and Outcome of Canadian Patients Diagnosed With Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is an extremely rare, heterogeneous disease of uncontrolled activation of the alternative complement pathway that is difficult to diagnose. We have evaluated the Canadian patients enrolled in the Global aHUS Registry to provide a Canadian perspective regarding the diagnosis and management of aHUS and the specific challenges faced. OBJECTIVE: To evaluate Canadian patients enrolled in the Global aHUS Registry to provide a Canadian perspective regarding the diagnosis and management of aHUS and the specific challenges faced. METHODS: The Global aHUS Registry is an observational, noninterventional, multicenter study that has prospectively and retrospectively collected data from patients of all ages with an investigator-made clinical diagnosis of aHUS, irrespective of treatment. Patients of all ages with a clinical diagnosis of aHUS were eligible and invited for enrollment, and those with evidence of Shiga toxin-producing Escherichia coli infection, or with ADAMTS13 activity ≤10%, or a subsequent diagnosis of thrombotic thrombocytopenic purpura were excluded. Data were collected at enrollment and every 6 months thereafter and were analyzed descriptively for categorical and continuous variables. End-stage renal disease (ESRD)-free survival was evaluated using Kaplan-Meier estimates, and ESRD-associated risk factors of interest were assessed using Cox proportional hazards regression models. Patients were censored at start of eculizumab for any outcome measures. RESULTS: A total of 37 Canadian patients were enrolled (15 pediatric and 22 adult patients) between February 2014 and May 2017; the median age at initial aHUS presentation was 25.9 (interquartile range = 6.7-51.7) years; 62.2% were female and 94.6% had no family history of aHUS. Over three-quarters of patients (78.4%) had no conclusive genetic or anti-complement factor H (CFH) antibody information available, and most patients (94%) had no reported precipitating factors prior to aHUS diagnosis. Nine patients (8 adults and 1 child) experienced ESRD prior to the study. After initial presentation, there appears to be a trend that children are less likely to experience ESRD than adults, with 5-year ESRD-free survival of 93 and 56% (P = .05) in children and adults, respectively. Enrolling physicians reported renal manifestations in all patients at initial presentation, and 68.4% of patients during the chronic phase (study entry ≥6 months after initial presentation). Likewise, extrarenal manifestations also occurred in more patients during the initial presenting phase than the chronic phase, particularly for gastrointestinal (61.1% vs 15.8%) and central nervous system sites (38.9% vs 5.3%). Fewer children than adults experienced gastrointestinal manifestations (50.0% vs 70.0%), but more children than adults experienced pulmonary manifestations (37.5% vs 10.0%). CONCLUSIONS: This evaluation provides insight into the diagnosis and management of aHUS in Canadian patients and the challenges faced. More genetic or anti-CFH antibody testing is needed to improve the diagnosis of aHUS, and the management of children and adults needs to consider several factors such as the risk of progression to ESRD is based on age (more likely in adults), and that the location of extrarenal manifestations differs in children and adults.

CONTEXTE: Le syndrome hémolytique et urémique atypique (SHUa) se caractérise par l'activation incontrôlée de la voie alternative du complément. Il s'agit d'une maladie rare, hétérogène et très difficile à diagnostiquer. Nous avons évalué les patients Canadiens inscrits au registre international du SHUa afin d'offrir une perspective canadienne sur le diagnostic et la prise en charge du SHUa, de même que sur les défis posés par la maladie. OBJECTIF: Évaluer les patients Canadiens inscrits au registre international du SHUa afin d'offrir une perspective canadienne sur le diagnostic et la prise en charge du SHUa, de même que sur les défis posés par la maladie. MÉTHODOLOGIE: Le registre international du SHUa est une étude observationnelle, non interventionnelle et multicentrique ayant recueilli, de façon rétrospective et prospective, des données auprès de patients de tous âges ayant reçu un diagnostic clinique de SHUa, quel que soit le traitement. Tous ces patients étaient admissibles et ont été invités à participer à l'étude. Les patients présentant une infection diagnostiquée à Escherichia coli producteur de shigatoxine, une activité de l'ADAMTS13 inférieure ou égale à 10 % ou un diagnostic subséquent de purpura thrombocytopénique thrombotique ont été exclus. Les données colligées à l'inclusion et à tous les six mois par la suite ont fait l'objet d'une analyze descriptive des variables catégorielles et continues. Des estimations de Kaplan-Meier ont été employées pour évaluer la survie sans insuffisance rénale terminale (IRT) et des modèles de régression à risques proportionnels de Cox ont servi à évaluer les facteurs de risques associés à l'IRT. Les patients ont été censurés au début du traitement par l'eculizumab pour la mesure des résultats. RÉSULTATS: Au total, 37 patients canadiens ont été inscrits (15 enfants et 22 adultes) entre février 2014 et mai 2017. L'âge médian lors de l'épisode initial était de 25,9 ans (intervalle interquartile: 6,7­51,7); 62,2 % des sujets étaient de sexe féminin et 94,6 % n'avaient pas d'antécédents familiaux de SHUa. Plus des trois quarts des patients (78,4 %) ne disposaient d'aucune information génétique ou relative aux anticorps anti-complément du facteur H concluante, et aucun facteur précipitant n'avait été rapporté avant le diagnostic pour la majorité des patients (94 %). Neuf patients (8 adultes et 1 enfant) avaient souffert d'IRT avant l'étude. Une tendance semble indiquer qu'après l'épisode initial, les enfants seraient moins susceptibles que les adultes de progresser vers l'IRT (survie sans IRT après 5 ans: 93 % et 56 % respectivement; P = 0,05). Les médecins-recruteurs ont observé des manifestations rénales chez tous les patients lors de l'épisode initial de SHUa et chez 68,4 % des patients au cours de la phase chronique (inscription à l'étude au moins 6 mois après l'épisode initial). Parallèlement, les manifestations extra-rénales sont également survenues chez davantage de patients lors de l'épisode initial que lors de la phase chronique, particulièrement pour les manifestations gastro-intestinales (61,1 % contre 15,8 %) et du système nerveux central (38,9 % contre 5,3 %). Les enfants ont été moins nombreux que les adultes à subir des manifestations gastro-intestinales (50,0 % contre 70,0 %), mais ont subi davantage de manifestations pulmonaires (37,5 % contre 10,0 %). CONCLUSION: Cette étude offre un éclairage sur le diagnostic et la prise en charge du SHUa chez les patients canadiens, de même que sur les défis posés par la maladie. Davantage de dépistage génétique et de dépistage des anticorps anti-CFH sont requis pour améliorer le diagnostic du SHUa. La prise en charge de la maladie doit tenir compte de plusieurs facteurs, notamment du risque de progression vers l'IRT qui varie selon l'âge (plus probable chez l'adulte) et du fait que le site des manifestations extrarénales diffère chez l'enfant et l'adulte.

Exploiting the vitamin B12 pathway to enhance oral drug delivery via polymeric micelles.

Vitamin B12 (VB12)-modified dextran-g-polyethyleneoxide cetyl ether (DEX-g-PEO-C16) was synthesized by linking VB12 residues to a DEX-g-PEO-C16 copolymer via a 2,2'-(ethylenedioxy)bis(ethylamine) spacer. The level of VB12 substitution on the DEX-g-PEO-C16 copolymer reached 1.68% (w/w). In aqueous solution, DEX-based copolymers form micelles that can entrap within their hydrophobic core up to 8.5% w/w of cyclosporin A (CsA), a poorly water soluble immunosuppressant. The permeability of Caco-2 cell membranes to CsA incorporated in VB12 modified and unmodified polymeric micelles was monitored in the presence and absence of intrinsic factor (IF). The apical (AP) to basolateral (BL) permeation of CsA through Caco-2 cell monolayers after 24 h of transport was significantly higher (1.8 and 2.3 times in absence and presence of IF, respectively) in the case of CsA loaded in VB12-modified polymeric micelles, compared to CsA in unmodified micelles. The results point to possible improvement in the application of polysaccharide-based polymeric micelles as targeted polymeric drug carriers for the oral delivery of poorly water soluble drugs.

Engineering polysaccharide-based polymeric micelles to enhance permeability of cyclosporin A across Caco-2 cells.

PURPOSE: To assess and compare the effectiveness of two types of polysaccharide-based micelles as delivery vehicles for poorly water soluble drugs by monitoring their permeability across Caco-2 cell monolayers. METHODS: Dextran (DEX) and hydroxypropylcellulose (HPC) were hydrophobically modified (HM) by grafting polyoxyethylene cetyl ether (POE-C16, 15 mol% and 5.4 mol%, respectively). The onset of micellization and mean diameter of polymeric micelles formed by HM-DEX and HM-HPC were determined by fluorescence spectroscopy and dynamic light scattering, respectively. Cyclosporin A (CsA)-loaded polymeric micelles were prepared by a dialysis procedure, and the amount of incorporated CsA was assayed by high performance liquid chromatography (HPLC). The stability of micelles in simulated gastric and intestinal fluids was studied as a function of contact time, and their cytotoxicity toward Caco-2 cells was evaluated using the MTT colorimetric assay. The bidirectional transport across Caco-2 cell monolayers of CsA entrapped in HM-DEX and HM-HPC micelles and of the polymers themselves was evaluated in the presence and absence of P-glycoprotein inhibitor. RESULTS: The amount of CsA incorporated in HM-HPC and HM-DEX micelles reached 5.5 and 8.5% w/w, respectively (entrapment efficiency of 22% or more). The polymeric micelles exhibited high stability in gastric and intestinal fluids and no significant cytotoxicity toward Caco-2 cells. The apical to basal permeability of CsA across Caco-2 cells increased significantly when loaded in polymeric micelles compared to free CsA. CONCLUSIONS: Polysaccharide-based polymeric micelles are promising carriers for the oral delivery of poorly water soluble drugs. In vitro tests indicate that, overall, HM-HPC micelles are more effective compared to HM-DEX micelles.

Solubilization of poorly water soluble drugs in micelles of hydrophobically modified hydroxypropylcellulose copolymers.

The main objective of this study is to exploit the solubilizing potential of hydroxypropylcellulose-g-polyoxyethylene alkyl ether (HPC-g-(POE)(y)-C(n)) polymeric micelles towards poorly water soluble drugs in order to improve their oral bioavailability. Hydrophobically modified HPC graft copolymers of various compositions were synthesized by attaching hexadecyl or octadecyl residues to the hydrophilic HPC backbone via short POE linkers of different lengths. The onset of micellization was estimated by fluorescence spectroscopy. The hydrodynamic diameter of different HPC-g-(POE)(y)-C(n) micelles was evaluated by dynamic light scattering (DLS). Cyclosporin A (CsA), a poorly water soluble immunosuppressant, was selected as model drug. CsA-loaded HPC-g-(POE)(y)-C(n) micelles were prepared by a dialysis procedure and the amount of CsA incorporated in the micelles was assayed by high-performance liquid chromatography. Following 24-h incubation with human colon adenocarcinoma, Caco-2 cells, the cytotoxicity of various HPC-g-(POE)(y)-C(n) copolymers was evaluated using the MTT colorimetric assay and compared to those of unmodified HPC and free (POE)(y)-C(n). In aqueous solution, different HPC-g-(POE)(y)-C(n) copolymers formed polymeric micelles of low critical association concentrations (CAC) and micelle mean diameters ranging from 78 to 90 nm. CsA loading into HPC-g-(POE)(y)-C(n) polymeric micelles was significantly larger than in unmodified HPC. It increased with increasing number of (POE)(y)-C(n) units grafted per HPC chain. On the cellular level, unmodified HPC showed no cytotoxicity, while free (POE)(y)-C(n) molecules inhibited cell growth. Most importantly, the study revealed that HPC-g-(POE)(y)-C(n) exhibited no significant cytotoxic effect.

Solubilization of cyclosporin A in dextran-g-polyethyleneglycolalkyl ether polymeric micelles.

Solubilization of the poorly water-soluble drug, Cyclosporin A (CsA), in aqueous dispersions of dextran-grafted-polyethyleneglycolalkyl ether (DEX-g-PEG-Cn) polymeric micelles was examined as a function of copolymer structure. In aqueous solution, DEX-g-PEG-Cn form polymeric micelles of low critical association concentrations (CAC) and small micelle sizes as determined by fluorescence spectroscopy and dynamic light scattering (DLS). Copolymers with longer polysaccharide chain showed larger CAC and mean diameter. The percentage of CsA loading into micelles was determined by high performance liquid chromatography. It was significantly larger in polymeric micelles compared to unmodified dextrans. It increased with increasing number of PEG-Cn units grafted per dextran chain and decreasing dextran molecular weight. The cytotoxicity of DEX-g-PEG-C(16) polymeric micelles towards Caco-2 cells, tested by MTT cytotoxicity assay, was significantly lower than that of free PEG-C(16) molecules. It can be concluded that the length of the hydrophilic part as well as the content and chemical nature of the hydrophobic substituents have an important effect on the ability of polymeric micelles to solubilize poorly-water soluble drugs.

Polymer based pH-sensitive carriers as a means to improve the cytoplasmic delivery of drugs.

pH-sensitive niosomal and liposomal formulations bearing alkylated N-isopropylacrylamide (NIPAM) copolymers were characterized with regard to vesicle-polymer interaction, pH-responsiveness and stability in human serum. The interactions between the pH-sensitive NIPAM copolymer and the vesicles were studied by spectrofluorimetry, using covalently-attached pyrene as a probe. In contrast to liposomes, where complexation of copolymer to the lipid bilayer is essentially mediated by hydrophobic interactions, the binding between niosomes and PNIPAM was mainly driven by hydrogen bonding. Both formulations were found to rapidly release their contents under mildly acidic conditions. However, the niosomes lost their pH-sensitivity after incubation in serum, whereas liposomes maintained their ability to respond to pH only when complexed with a copolymer containing a high proportion of hydrophobic anchor. The ability of pH-sensitive liposome/polymer complexes to enhance the cytotoxicity of cytosine arabinofuranoside (ara-C) was evaluated in vitro using macrophage-like J774 cells. Ara-C encapsulated in pH-sensitive liposomes exhibited a higher cytotoxicity than the control formulation. This study showed that both niosomes and liposomes can be rendered pH-sensitive by anchoring a randomly-alkylated NIPAM copolymer to their surface. The interactions that take place between the polymer and the vesicles strongly depend on the vesicle nature. pH-sensitive PNIPAM-based liposomes can improve the in vitro efficiency of ara-C.

Preparation and tumor cell uptake of poly(N-isopropylacrylamide) folate conjugates.

Folate conjugates (PNIPAM-NH-FA) of a copolymer of N-isopropylacrylamide (NIPAM) and amino-N'-ethylenedioxy-bis(ethylacrylamide) were prepared by an efficient synthesis leading to random grafting, via a short dioxyethylene spacer, of approximately 7 folic acid residues per macromolecule. The chemical composition of the copolymer was characterized by (1)H NMR and UV/vis spectroscopy. A fluorophore-labeled folate PNIPAM conjugate was tested by in vitro assays performed with cultured KB-31 cells overexpressing the folate receptor. The cellular uptake of the copolymer was found to be temperature dependent and was competitively decreased by free folic acid, indicating that the polymer uptake is mediated specifically by the folate receptor. Hydrophobically modified folate conjugates of NIPAM, amino-N'-ethylenedioxy-bis(ethylacrylamide) copolymers, bearing a small number of n-octadecyl groups were prepared following a modified synthetic procedure for use in future studies of FA-targeted liposomes.