Enzyme replacement therapy in a feline model of MPS VI: modification of enzyme structure and dose frequency.

Enzyme replacement therapy (ERT) in the MPS VI cat is effective at reducing or eliminating pathology in most connective tissues. One exception is that cartilage and chondrocytes remained distended with extensive lysosomal vacuolation after long-term, high-dose ERT. In this study, we demonstrate that recombinant human N-acetylgalactosamine-4-sulphatase (4S) is taken up by chondrocytes via a mannose-6-phosphate-dependent mechanism and is effective at removing MPS storage. In vitro, the penetration of 4S into articular cartilage is low (partitioning coefficient = 0.06) and i.v. administered enzyme does not distribute significantly into articular cartilage in vivo. To alter the tissue distribution of 4S, the enzyme was coupled to ethylene diamine or poly-L-lysine, increasing its overall charge and diffusion into cartilage, and the dosing frequency of unmodified 4S was increased. Modification resulted in active 4S that maintained its ability to correct MPS storage and increased the partitioning coefficient of 4S into cartilage by 77% and 50% for ethylene diamine and poly-L-lysine, respectively. However, in vivo ERT studies demonstrated that response to therapy was not significantly improved by either the enzyme modifications or change to the dosing regimen, when compared with ERT with unmodified enzyme. Distribution experiments indicated the majority of enzyme is taken up by the liver irrespective of modification. To optimize therapy and improve the amount of enzyme reaching cartilage and other tissues demonstrating poor uptake, it may be necessary to bypass the liver or prolong plasma half-life so that proportionately more enzyme is delivered to other tissues.

Histomorphometric analysis of the tibial growth plate in a feline model of mucopolysaccharidosis type VI.

Mucopolysaccharidosis type VI (MPS VI) is a genetically inherited lysosomal storage disorder. Severely affected children exhibit a range of skeletal abnormalities including short stature, facial dysmorphia, and dysostosis multiplex. Naturally occurring and transgenic animal models of MPS VI are also found which exhibit pathology similar to the human disorder. In this paper we have characterized the formation of trabecular bone from growth plate cartilage in a feline model of MPS VI. Tibial trabecular bone was shown to be osteopenic in MPS VI animals with a bone mineral volume (BV/TV) of 4.51% compared with a BV/TV of 15.64% in normal animals. In addition to osteopenia, a rearrangement of trabecular bone architecture was also observed in MPS VI tibiae, with fewer, thinner trabeculae noted; bone formation rate was also decreased. These observations support those previously made in the L5 vertebrae of MPS VI animals. When the sequential formation of growth plate cartilage structural elements, their transition into primary bone spongiosa, and remodeling into secondary bone spongiosa was characterized, no difference between normal and MPS VI could be detected in the number of cartilage septae and their arrangement in the proliferative and hypertrophic regions of the growth plate or trabecular elements in the primary spongiosa. However, a deviation from normal was observed in the resting zone of the growth plate and in the secondary spongiosa of bone. Thus, the osteopenia observed in MPS VI bone appears to arise primarily from a defect in bone production within the metaphysis and diaphysis rather than the creation of an abnormal template in the preceding growth plate cartilage.

Glycosaminoglycan accumulation and excretion in the mucopolysaccharidoses: characterization and basis of a diagnostic test for MPS.

A combination of anion-exchange chromatography and 30-40% gradient polyacrylamide gel electrophoresis (gradient-PAGE) was used to purify and characterize urinary glycosaminoglycans from various mucopolysaccharidoses (MPS). The urinary glycosaminoglycans from the different MPS displayed distinct patterns on gradient-PAGE and further confirmation of MPS types and subtypes was demonstrated by an electrophoretic shift in the banding pattern after digestion with the appropriate MPS enzyme. Thus each of the MPS accumulates a unique spectrum of glycosaminoglycans with a nonreducing terminal consisting of the substrate specific for the deficient enzyme in that particular MPS disorder. The absolute correlation of the nonreducing terminal structure with a particular MPS and the availability of recombinant lysosomal enzymes provide the means for a rapid and accurate diagnosis of individual MPS. Analysis of tissue glycosaminoglycans in one MPS type (feline MPS VI) indicated a tissue-specific pattern of glycosaminoglycan accumulation. Undegraded glycosaminoglycans had distinct banding patterns on gradient-PAGE and although dermatan sulfate was predominantly excreted in MPS VI urine, some tissues were observed to accumulate predominantly chondroitin sulfate glycosaminoglycans, e.g., bone and kidney. The spectrum of glycosaminoglycans excreted in the urine is therefore most likely a combination of glycosaminoglycans from various tissues.

Recombinant caprine 3H-[N-acetylglucosamine-6-sulfatase] and human 3H-[N-acetylgalactosamine-4-sulfatase]: plasma clearance, tissue distribution, and cellular uptake in the rat.

The use of recombinant lysosomal enzymes for enzyme replacement therapy (ERT) is likely to be a necessary component of effective treatment regimens for lysosomal storage diseases (LSDs). The mechanism and rate of uptake into target cells, rate of disappearance of the enzyme from plasma, and its tissue distribution are important factors to assess the need for possible modifications to the enzyme, particularly for LSDs that affect the central nervous system (CNS). Two recombinant lysosomal enzymes, caprine N-acetylglucosamine-6-sulfatase (rc6S) and human N-acetylgalactosamine-4-sulfatase (rh4S), deficient in MPS IIID and MPS VI, respectively, were radiolabeled and purified. The major portion (>77%) of each recombinant enzyme contained the mannose-6-phosphate (M6P) recognition marker as demonstrated by their ability to bind to a M6P receptor affinity column. The uptake of 3H-rc6S and 3H-rh4S into cultured rat brain cells was also inhibited by the addition of 5 mM M6P to the culture medium. After iv administration of 0.4-0.5 mg/kg of 3H-rc6S and 1 mg/kg of 3H-rh4S to the rat, both enzymes were rapidly lost from the circulation in a biphasic fashion (t1/2 for 3H-rc6S = 1.25+/-0.15 min and 37.17+/-23.29 min; t1/2 for 3H-rh4S = 0.41 and 5.3 min). At this dose, about 6% of 3H-rc6S, but only 0.49% of 3H-rh4S, remained in the plasma 4 h after administration, whereas approx 30% of 3H-rc6S and more than 50% of 3H-rh4S was found in the liver. At doses of 1.6-2.0 mg/kg of 3H-rc6S and 1 mg/kg 3H-rh4S, but not at the lower dose of 3H-rc6S, trace levels of both 3H-rc6S and 3H-rh4S were detected in the brain. The low level of enzyme recovered from the brain suggests that modification of rc6S will be necessary to achieve sufficient enzyme uptake into the CNS for effective therapy of MPS IIID.