Urinary glycosaminoglycans as a potential biomarker for evaluating treatment efficacy in subjects with mucopolysaccharidoses

Molecular Genetics and Metabolism 130 (2020) 7–15

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Review article

Urinary glycosaminoglycans as a potential biomarker for evaluating treatment efficacy in subjects with mucopolysaccharidoses

T

⁎

Emil Kakkis , Deborah Marsden Ultragenyx Pharmaceutical Inc., Novato, CA, United States of America

A R T I C LE I N FO

A B S T R A C T

Keywords: Biomarker Enzyme replacement therapy Glycosaminoglycan Mucopolysaccharidoses

Accumulations of glycosaminoglycans (GAGs) that result from deficiencies in lysosomal hydrolases are characteristic of mucopolysaccharidoses (MPS). Enzyme replacement therapies (ERTs) are now available for several MPS diseases (MPS I, MPS II, MPS IVA, MPS VI, and MPS VII), but assessment of the efficacy of treatment can be challenging because these are rare, progressive, and highly heterogeneous diseases; because some clinical manifestations may be irreversible if treatment initiation is delayed; and because determining the benefits of a treatment to prevent those manifestations may take prolonged periods of time. In addition to accumulation of GAGs in tissues, elevated urinary GAG (uGAG) levels are evident and are reduced rapidly after initiation of ERT. Studies in MPS animal models and clinical studies in subjects with MPS diseases have revealed correlations between reductions of uGAG levels and clinical effects of ERTs. In this article, we review the growing body of evidence to support the potential for the use of uGAG levels as predictive biomarkers of treatment efficacy.

1. Introduction Mucopolysaccharidoses (MPS) are rare, progressive disorders caused by genetic defects in one of 11 lysosomal hydrolases required for degradation of glycosaminoglycans (GAGs) [1,2]. The resulting accumulation of GAGs in multiple tissues causes extensive damage that can vary according to the affected hydrolase enzyme and nature of the defect. Enzyme replacement therapy (ERT) by intravenous infusion of a functional version of the defective enzyme has proven to be an effective mean to treat several MPS diseases, including MPS I [3], MPS II [4], MPS IVA [5], MPS VI [6], and most recently MPS VII [7]. By directly targeting the underlying defect and reversing the biochemical block, ERT reduces the accumulation of tissue GAGs, thereby preventing tissue damage and potentially allowing some reversal of existing damage. However, assessing the clinical benefits of treatment for MPS diseases can be challenging. Some tissue damage prior to diagnosis and treatment may be irreversible even after restoration of enzyme activity, and determination of benefits of a treatment to prevent progression of damage can take prolonged periods, particularly for rare diseases for which the number of subjects is small. Therefore, there is a need for reliable, predictive markers of treatment efficacy. The accumulation of GAGs in tissues of patients with MPS diseases

also result in elevated urinary GAG (uGAG) levels. uGAG levels have been found to be a useful marker for the efficacy of ERTs because they are readily measured without invasive procedures, rapidly reduced after initiation of treatment, responsive to changes in dosing, and reflect restoration of enzyme activity in affected tissues (Table S1). Renal tubules are the likely source for large molecular weight uGAGs, which are measured using dimethylene blue (DMB) or alcian blue dye-based binding methods [8]. Studies in the MPS I canine model [9,10] showed that at low doses of iduronidase, the glomeruli are cleared but not the tubules; at the higher doses, the tubules are cleared as well as the uGAG. Given the methods preferentially detect high molecular weight highly anionic GAG with limited ability to be filtered by the glomerulus and the correlation of the tubular storage with uGAG in MPS I dogs, the majority of uGAG is believed to be derived from tubule cells that are heavily engorged with GAG on histology. Specialized biochemical genetics clinical laboratories that utilize liquid tandem mass spectrometry (LC-MS/MS) increasingly also offer sensitive uGAG measurement via an LC-MS/MS platform, of which there are multiple methods reported, and these methods can detect very small fragments of GAG, some of which have a greater potential to be filtered [11–13]. Most LC-MS/MS methods rely broadly on either chemical or enzymatic digestion of GAGs prior to measurement; however, one variation of LC-MS/MS

Abbreviations: 6MWT, 6-Minute Walk Test; DMB, dimethylene blue; ERT, enzyme replacement therapy; FVC, forced vital capacity; GAG, glycosaminoglycan; KS, keratan sulfate; LC-MS/MS, liquid tandem mass spectrometry; MID, minimally important difference; MPS, mucopolysaccharidoses; QOW, once every other week; rhASB, rh-arylsulfatase B; rhIDU, recombinant human iduronidase; uGAG, urinary glycosaminoglycan ⁎ Corresponding author at: 60 Leveroni Court, Novato, CA 94949, United States of America. E-mail address: [email protected] (E. Kakkis). https://doi.org/10.1016/j.ymgme.2020.02.006 Received 9 December 2019; Received in revised form 10 February 2020; Accepted 17 February 2020 Available online 19 February 2020 1096-7192/ © 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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spleen, lymph nodes, kidney, and lung. A general dose response was observed in which increasing the dose level of rhIDU corresponded with a greater reduction in tissue GAG levels, with immune-tolerant canines achieving greater tissue GAG reduction than nontolerant canines per dose level. Reduction in uGAG levels corresponded with tissue GAG reductions, with canines with MPS I that were not tolerant to rhIDU demonstrating an uGAG level reduction of approximately 60 to 66% (although with induced rhIDU tolerance, uGAG level reduction improved to 86–93%). Moreover, a similar trend was observed in the histopathology of these canines with immune tolerance and higher dose levels [18]. Together, these results indicate that uGAG levels are indicative of tissue GAG levels and underlying tissue histopathology in MPS I. These results also indicate that uGAG levels are sensitive to biological factors that might hinder enzyme function, such as antibodies, and can detect in finely graded pattern differences in tissue GAG levels in the most difficult-to-treat tissues. Although a precise correlation between the tissues of canines and humans with MPS I is not possible, reductions in uGAG lead to substantial reductions in tissue pathology in animals (in some cases to normal or near-normal appearance) and uGAG reductions were observed at the same dose in human studies demonstrating clinical benefit and providing a reasonable confirmation that results from canine studies are relevant to assessing the effects of ERT in humans. As discussed below, clinical study data corroborate this finding that uGAG reduction is directly associated with clinical benefit.

measures endogenous GAG oligosaccharides without the need for prior digestion [12]. The LC-MS/MS platform allows for increased sensitivity and specificity to precisely measure GAGs, albeit at an increased cost to perform. To assess whether uGAG can accurately represent the disease status within diverse relevant tissues and reflect the treatment effect, establishment of a relationship between the quantity of reduction in uGAG excretion and the improvement in tissue pathology or storage is important. This relationship is not easily demonstrated in humans given the inability to access all tissues, but is more tractable in animal models. As explained in the sections that follow, studies in multiple animal models of MPS diseases have shown that changes in uGAG levels correlate with and predict changes in both tissue GAG levels and tissue pathology, and that uGAG best reflects the more difficult-to-treat tissues. In clinical studies, growing evidence also supports correlations between uGAG excretion and meaningful benefits. In this manuscript, we review the available preclinical and clinical evidence to support the use of uGAG levels as a predictive biochemical marker of ERT efficacy. 1.1. MPS I: observational and preclinical evidence MPS I (Hurler syndrome, Hurler-Scheie syndrome, or Scheie syndrome) is caused by defects in lysosomal iduronidase. Manifestations include hepatosplenomegaly, coarse facies, hernias, joint stiffness, pulmonary insufficiency, heart disease, skeletal dysplasia, and progressive intellectual disability [14]. Clinical studies of MPS I have shown that uGAG levels correlate with severity of disease. As shown in Fig. 1, the patients with the most severe type of MPS I (Hurler syndrome) typically have uGAG levels in the range of 300 to 600 μg/mg creatinine, whereas those with the intermediate type of MPS I (HurlerScheie syndrome) have uGAG levels in the range of 100 to 300 μg/mg creatinine; our own data indicate that patients with attenuated Scheie syndrome have levels less than 100 μg/mg creatinine [3,15,16]. Multiple studies have demonstrated that uGAG levels accurately reflect tissue GAG storage and pathology in MPS I canines undergoing ERT [9,10,17]. The largest such study used recombinant human iduronidase (rhIDU) in 24 canines with MPS I [18]; two dose levels of rhIDU were administered to canines with varying degrees of induced immune tolerance. In this study of ERT, immune-tolerized canines demonstrated decreased tissue GAG levels, lysosomal pathology, and uGAG levels, indicative of effective enzyme replacement. Reduction in tissue GAG levels was observed across a range of sample tissues, including the liver,

1.2. MPS I: clinical study evidence An initial Phase 1/2 trial of laronidase in MPS I showed that uGAG was reduced rapidly within a few weeks of initiating therapy and was associated with clinical benefits observed later over time in an openlabel study [3]. Further follow-up of these subjects showed that uGAG levels further reduced over a 6-year period, and that the surviving six patients who had done well had no antibodies and excellent uGAG reduction [19]. The laronidase Phase 3 study provided clinical support for the validity of uGAG in predicting clinical effect. In this study, 45 subjects with MPS I were randomized to 0.58 mg/kg weekly or placebo and were assessed over a 26-week period for changes in the 6-Minute Walk Test (6MWT) and forced vital capacity (FVC) as co-primary endpoints [16]. All 22 subjects treated with active therapy had a reduction in uGAG levels (mean 54.1% reduction). In the 6MWT, 41% of subjects taking laronidase achieved a minimally important difference (MID) of 54-m increase (as defined for chronic obstructive pulmonary disease subjects [20]; no MID for MPS has been defined) versus only 11% of placebo subjects (p = .047). Similarly for FVC, 41% of subjects treated with laronidase achieved a clinically meaningful increase in FVC of 11% versus 9% for placebo (p = .016). Longer-term observation of this cohort over 3 years further supported that those subjects with sustained GAG reduction had good long-term impact on MPS I disease [21]. An additional study in MPS I subjects under age 5 years assessed how antibodies might interfere with efficacy [15]. In the study, subjects with null genotypes (n = 4) developed high-titer antibodies and had uGAG levels reduced by less than 50%. Per protocol, the enzyme dose was doubled for these subjects, and resulted in significant further improvement in uGAG reduction (more than 50%). This provides some evidence that insufficient uGAG reduction could be overcome by a higher dose. When correlated with canine MPS I data and the impact of antibodies on tissue, it is also clear that uGAG can measure decreases in efficacy due to anti-iduronidase antibodies and that increased dose can improve the tissue outcome.

Fig. 1. Correlation of Clinical Phenotype With uGAG Excretion Level. The number of subjects in each study are shown in the bars. Of the 45 subjects in the Phase 3 study by Wraith [16], 38 had Hurler-Scheie syndrome and seven had Scheie syndrome. GAG, glycosaminoglycan; MPS, mucopolysaccharidoses; uGAG, urinary glycosaminoglycan.

1.3. MPS II: preclinical evidence MPS II (also called Hunter syndrome) is an X-linked disorder caused by defects in the lysosomal iduronate-2-sulfatase enzyme. Most affected 8

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Fig. 2. Animal Model of Tissue GAG Levels in MPS II Mice After 5 Weekly Doses of Idursulfase Enzyme Replacement Therapy. Source: [23]. *significantly different from vehicle control at P < 0.05. Values shown are ± SEM. GAG, glycosaminoglycan; MPS, mucopolysaccharidoses; SEM, standard error of the mean; WT, wild-type.

individuals are male and may present with early progressive disease (accompanied by progressive cognitive decline and cardiac disease) or slowly progressive disease; both forms include short stature, macrocephaly, macroglossia, hearing loss, hepato-splenomegaly, and spinal stenosis [22]. In the mouse model of MPS II, dose-ranging studies with ERT demonstrated that elevated uGAG levels responded to treatment in a dosedependent manner [23]. Reduction in uGAG levels over the dose range of 0 to 1 mg/kg adequately reflected GAG reduction in a variety of tissues, including the liver, spleen, heart, and kidney (Fig. 2). As expected, the liver and spleen demonstrated marked reductions in tissue GAG levels with all doses of the drug, whereas levels in the lung were more resistant to treatment. Tissue GAG levels in the kidney tended to show a dose-proportionate response [23]. Although not directly assessed, given the relevance of the kidney to uGAG levels, this strongly suggests that uGAG levels are also likely to be dose proportionate in this animal model.

Fig. 3. Correlation of Clinical Outcomes With uGAG Levels Over Time. Correlation of (A) 6MWT, (B) uGAG, and (C) FVC by weeks of idursulfase ERT. Source: [4]. *P=0.0011 compared with placebo and P=0.0176 compared with EOW idursulfase at Week 53. 6MWT, 6-Minute Walk Test; EOW; every-otherweek; ERT, enzyme replacement therapy; FVC, forced vital capacity; GAG, glycosaminoglycan; SEM, standard error of the mean; uGAG, urinary glycosaminoglycan.

1.4. MPS II: clinical evidence In the idursulfase development program, uGAG levels have also demonstrated an ability to discriminate between effective and less effective dose regimens. In the Phase 1 randomized controlled study, 12 subjects were treated with idursulfase ERT at three dose levels (low 0.15, mid 0.5, and high 1.5 mg/kg every other week [QOW] for 24 weeks followed by an open-label extension study). After 6 months of treatment, uGAG levels had decreased by 41 to 58% at all three dose levels, although no substantial changes in 6MWT were observed in any of the treatment groups. After 12 months of treatment, reductions in uGAG levels were sustained. However, improvement in 6MWT only occurred at the two highest dose levels, with the largest improvement in 6MWT at the 1.5 mg/kg dose (the only dose to achieve uGAG reduction of more than 50%) [24]. Similar patterns of uGAG preceding and differentiating clinical effects were observed in the Phase 3 randomized controlled study of idursulfase ERT. In this study, 96 subjects were treated with idursulfase at two dose levels (0.5 mg/kg QOW and 0.5 mg/kg weekly) or with placebo over a 1-year period [4]. uGAG levels and the primary endpoints of 6MWT and FVC were measured at Weeks 18, 36, and 53. By Week 18, maximum uGAG reductions had been achieved for each dose, but clinical efficacy was not yet observed (Fig. 3). At Week 53, uGAG reduction was sustained at 58.3% in the weekly dose and 43.9% in the QOW dose, with corresponding dose-related clinical effects for both FVC (weekly vs QOW: +0.22 vs 0.07 L; p = .0131) and 6MWT (weekly vs QOW: 44.3 vs 30.3 m; p = .001). Thus, the maximal reductions in

uGAG achieved by Week 18 predicted the extent of clinical benefit at Week 53.

1.5. MPS IVA: clinical evidence MPS IVA (also called Morquio A syndrome) is caused by a deficiency in lysosomal N-acetylgalactosamine-6-sulfatase. Clinical manifestations include skeletal and connective tissue abnormalities; in addition, and pulmonary and cardiac manifestations are common [25]. Although there was no MPS IVA animal model for study, ERT development work in subjects with MPS IVA has shown the predictive value of uGAG on clinical outcomes. In this program, the uGAG studied was keratan sulfate (KS; assessed using LC-MS/MS analysis of two KS mono- and di-sulfated disaccharides), which is excreted in relatively smaller quantities in affected subjects [26,27]. The Phase 1/2 MOR-002 trial was a dose titration study conducted in 20 subjects with MPS IVA aged 5 to 18 years [28]. Over a 36-week period, subjects received 12 weeks of elosulfase alfa 0.1, 1.0, or 2.0 mg/kg elosulfase alfa, after which all doses were adjusted to 1.0 mg/kg. In the 0.1 mg/kg group, the 6MWT results improved only after the dose increase (16.3 m). When 9

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the 2.0 mg/kg was ramped down, improvement decreased from 13.8 to 4.0 m. When uGAG was analyzed, uGAG decreased substantially only beginning at 1.0 mg/kg and was maximally reduced at 2.0 mg/kg. After the dose reduction to 1.0 mg/kg, uGAG KS levels increased, indicating that 2.0 mg/kg weekly was a required dose for optimal uGAG reduction. The Phase 3 MOR-004 trial was a randomized, double-blind, placebo-controlled study in 176 subjects that examined weekly or QOW doses of elosulfase alfa 2.0 mg/kg [28]. The study showed that the weekly dose regimen had a modeled treatment effect size on the 6MWT of 22.5 m (p = .0174), whereas the QOW regimen did not show a statistically significant effect. When evaluating urinary KS, the two regimens showed a substantial difference in reduction. After 24 weeks, reductions of approximately 45 to 50% were evident in the weekly treatment group, whereas reductions were only approximately 30% in the QOW treatment group, demonstrating that uGAG reduction was predictive for the optimal clinical dose as assessed by the 6MWT [28]. These results in MPS IVA reinforce that reductions of uGAG excretion of more than 50% are likely to represent the threshold associated with clinical benefit. Moreover, uGAG levels were responsive to differences in dose regimen and demonstrated substantially less inter-individual variability compared with clinical outcomes such as 6MWT. Thus, evaluation of uGAG levels potentially allows a more precise assessment of treatment effects both in groups and in individual subjects.

Fig. 4. Levels of uGAG Excretion Over Time in Subjects With MPS VI Receiving Galsulfase ERT in a Phase 1 Galsulfase Study. Each point is the mean of 3 subjects; therefore, no error bars are provided. Source: [34]. ERT, enzyme replacement therapy; MPS, mucopolysaccharidoses; uGAG, urinary glycosaminoglycan.

subjects with MPS VI, administration of 1 mg/kg rhASB led to significant improvements in endurance, as measured by the 12-Minute Walk Test (p = .025) and the 3-Minute Stair Climb (p = .053). Reduction in uGAG levels were statistically significant (p < .001) and reductions of more than 50% were achieved in 17 of 19 subjects on active therapy and none of the 20 subjects taking placebo (72% reduction relative to placebo) [6]. An extensive survey study of 121 MPS VI subjects was conducted to assess the relationship between uGAG, clinical phenotype, and longterm outcomes in subjects with MPS VI [35]. This study demonstrated that high uGAG levels were associated with an accelerated clinical course comprised of age-adjusted short stature and low body weight, impaired endurance, compromised pulmonary function, and reduced joint range of motion. There were clear negative correlations of uGAG levels with both 6MWT and shoulder range of motion (Fig. 5A, B). Higher uGAG levels also correlated with severe pulmonary restriction (Fig. 5C, D). Most subjects who survived for longer periods and reached age at least 24 years generally had less severe uGAG excretion and usually had less severe clinical disease. Only one individual older than age 22 years had levels of uGAG more than 100 μg/mg creatinine, suggesting that most individuals with high uGAG levels die early in life. The 10-year follow-up study of patients who previously participated in the survey study (Resurvey Study) evaluated medical histories and clinical assessments in 59 subjects (ERT treatment, n = 55; no treatment, n = 4) and survival status in 117 subjects older than age 12 years [36]. ERT-treated subjects received a mean (SD) of 6.8 (2.2) years of galsulfase ERT between baseline (original survey study) and follow-up. Greater increases in height were observed for subjects who initiated ERT between ages 4 to 7 years versus 8 to 12 years old. The greatest increases in FVC (68%) and forced expiratory volume (55%) were observed for subjects who initiated ERT at age less than 13 years. However, in subjects with baseline uGAG levels more than 200 μg/mg creatinine, FVC was increased by only 48% in the less than 13-year-old baseline age group and by only 15% in the > 13-year-old baseline age group. ERT-treated subjects who completed the 6MWT demonstrated a mean (SD) increase of 65.7 m (100.6 m). Observed mortality in untreated subjects was 50% (7/14) but was only 16.5% (17/103) in the ERT group (unadjusted hazard ratio, 0.24; 95% CI 0.10–0.59). KaplanMeier analyses suggested that probability of survival was greater for subjects with uGAG levels of up to 200 μg/mg creatinine. Reductions in uGAG levels over this interval were 87.9% for ERT-treated subjects and 49.8% for untreated subjects. Although the reduction in untreated subjects was considerable, the number of subjects was small (n = 4), and this might have reflected age-related decreases in uGAG and/or selection (survival) of only subjects whose levels spontaneously decreased. The most important new association was that a greater

1.6. MPS VI: preclinical evidence MPS VI (also called Maroteaux-Lamy syndrome) is caused by deficiency of lysosomal arylsufatase B. Manifestations of both severe and mild forms include skeletal and respiratory abnormalities, and the severe form may also be accompanied by hearing and vision impairment, as well as heart disease [29]. Preclinical data on uGAG in MPS VI are limited, but studies in a cat model again show a substantial dose-responsive reduction in uGAG that was associated with tissue delivery of rh-arylsulfatase B (rhASB) and dose-responsive improvements in lysosomal storage and clinical measures, including skeletal disease, when treatments were started at a young age [30–32]. For example, in a study of untreated normal and MPS VI control cats, uGAG levels in the MPS VI controls remained approximately 10-fold elevated above normal and ERT-treated MPS VI cats [32]. Maximum reductions in uGAG levels in ERT-treated cats were seen at 0.2 and 1 mg/kg, and the largest changes in tissue pathology were seen at 1 mg/kg, suggesting that a sustained, threshold reduction in GAGs (at least 50%) is necessary to produce significant changes in pathology [32]. Other studies have also shown dose-related reductions in skeletal disease and spinal cord compression in the cats with MPS VI when treated early in life [30,32,33]. Although the number of animals treated and the data published do not allow the extensive comparison demonstrated in the MPS I canine model, the MPS VI cat data are still supportive of the strong association of uGAG levels with tissue pathology and clinical benefit. 1.7. MPS VI: clinical evidence In the Phase 1/2 randomized, double-blind study of galsulfase ERT, seven subjects were randomized and six completed 24 weeks of weekly therapy of either low-dose 0.2 mg/kg therapy (n = 3) or higher-dose 1 mg/kg therapy (n = 3) [34]. The low-dose subjects reached a 51% reduction in uGAG levels as measured by a DMB-based uGAG assay method, whereas the higher-dose subjects achieved and maintained a 63% reduction (Fig. 4). One subject in the low-dose group withdrew from the study; the other low-dose subjects achieved 70% reduction in uGAG when they were initiated on the higher dose. Although the clinical results were inconclusive due to the small size of the study, the higher dose resulted in higher levels of circulating enzyme. During the 6-month, randomized, Phase 3 study of galsulfase ERT in 10

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E. Kakkis and D. Marsden

Fig. 5. Correlation of Levels of uGAG Levels With Clinical Outcomes for (A) 6MWT, (B) Range of Motion, and (C, D) FVC in Subjects With MPS VI. Source: [27]. 6MWT, Six-Minute Walk Test; FVC, forced vital capacity; uGAG, urinary glycosaminoglycan.

has profound systemic benefits on pathology in a variety of tissues and many important clinical measures [39–44]. However, uGAG excretion in MPS VII animal models has not been commonly or completely studied because obtaining urine in adequate quantities is more difficult in mice.

reduction in uGAG was associated with a substantial decrease in mortality in subjects older than age 12 years with a hazard ratio of 0.24. Fifteen-year survival data for patients from the initial survey study further show that galsulfase-treated patients (n = 104) continue to have a survival advantage over treatment-naïve patients (n = 14), with mortality rates of 24% versus 57%, respectively [37]. That study did not directly assess individual correlations between uGAG and outcomes, but the general association with uGAG reduction and long-term clinical outcomes and mortality were clear from the study. Given that the galsulfase randomized Phase 3 study showed a 72% reduction in uGAG after 6 months of treatment, the data support that uGAG reduction in this range is associated with a substantial reduction of about 75% in mortality risk. These data are consistent with those from the preclinical studies and demonstrate that uGAG reductions of more than 50% are associated with improvements in clinical effects, with further improvements at higher levels of uGAG reduction.

1.9. MPS VII: clinical studies There are no published historical data in subjects with MPS VII to establish a relationship between uGAG and disease severity because no clinical surveys were conducted in this population prior to the initiation of the ERT development program for vestronidase alfa (recombinant human β-glucuronidase). However, the data published from the Phase 3 clinical trial of vestronidase alfa also support the relationship between uGAG levels and clinical benefit in subjects with MPS VII [7]. In that randomized Blind-Start, crossover study, treatment efficacy was assessed in 12 subjects after 24 weeks of treatment preceded by placebo treatment periods of 0, 8, 16, or 24 weeks (groups 1–4, respectively). Excretion of the uGAG dermatan sulfate was significantly reduced within 2 weeks in all subjects and an overall 65% reduction was observed by 24 weeks (least squares mean change; p < .0001). On a composite endpoint of clinical benefit (Multi-Domain Responder Index; included 6MWT, FVC, shoulder flexion, visual acuity, and BruininksOseretsky Test of Motor Proficiency), 10 of 12 patients showed improvements in at least one domain that were considered clinically meaningful based on their use in other diseases (overall mean [SD] change +0.5 [0.8]). These data are consistent with an association of

1.8. MPS VII: preclinical studies MPS VII (also called Sly syndrome) is caused by a deficiency of lysosomal β-glucuronidase, frequently presents with hydrops fetalis at birth, and manifests with symptoms that may include abnormal coarsened facies, pulmonary disease, hepatosplenomegaly, short stature, cognitive impairment, and skeletal abnormalities [38]. This disorder is considered ultra-rare, with an estimated frequency of between 1:300,000 and 1:2,000,000 [38]. Several studies have demonstrated that ERT in the MPS VII mouse 11

Design

RDBPC (n = 45)

Open-label (n = 40)

Phase 3 [16]

Phase 3 extension [21]

12

Open-label (n = 94)

Phase 3 extension [45]

0.15 mg/kg QOW for 24 wks 0.5 mg/kg QOW for 24 wks 1.5 mg/kg QOW for 24 wks 0.5 mg/kg QOW for 53 wks 0.5 mg/kg QW for 53 wks 0.5 mg/kg QW for 3 yrs

0.58 mg/kg QW for 3.5 yrs

0.58 mg/kg QW for 26 wks

0.58 mg/kg QW for 6 yrs

0.58 mg/kg QW for 52 wks

Dose

RDBPC (n = 176)

Mucopolysaccharidosis disease: MPS VI Enzyme replacement therapy: galsulfase Phase 1/2 [34] RDBPC (n = 6)

Phase 3 [47]

0.2 mg/kg QW for 24 wks 1 mg/kg QW for 24 wks

2.0 mg/kg QOW or QW for 24 wks

Mucopolysaccharidosis disease: MPS IVA Enzyme replacement therapy: elosulfase alfa Phase 1/2 [46] Dose titration 0.1–2.0 mg/kg (n = 20) over 36 wks

RDBPC (n = 96)

Phase 3 [4]

Mucopolysaccharidosis disease: MPS II Enzyme replacement therapy: idursulfase Phase 1/2 [24] RDBPC followed by open-label (n = 12)

Open-label (n = 5)

Phase 1 extension [19]

Mucopolysaccharidosis disease: MPS I Enzyme replacement therapy: laronidase Phase 1 [3] Open-label (n = 10)

Development phase

24% 95%

−63%

2.0 mg/kg QOW: 0.5 m (p = .954) 2.0 mg/kg QW: 22.5 m (p = .017)

4 m at Week 72

−51%

ND

ND

−77.4%

44.3 m (p = .00131) Sustained improvement

17.1 m

−72%

−58.3%

38.1 m (p = .066) (ANCOVA p = .037)

−54.1%

6 mo: no change 12 mo: no change 6 mo: no change 12 mo: 10.9% 6 mo: no change 12 mo: 27.9% 30.3 m (p = .0732)

ND

−76.9%

6 mo: −41% 12 mo: −47% 6 mo: −44% 12 mo: −43% 6 mo: −58% 12 mo: −58% −43.9%

ND

Walk test

−63%

uGAG %

Table 1 Clinical study summary of enzyme replacement therapy for mucopolysaccharidosis diseases.

−14% pooled analysis

ND

ND

ND 2.0 mg/kg QOW: 3.0% from BL 2.0 mg/kg QW: 3.3% from BL

Improvement in 1 subject in pooled analysis

ND

Sustained improvement

ND

Sustained improvement

−25.1% (p < .0001)

12.5% at Week 72

No change

−25.3% (p < .0001)

−19.8% (p < .0001)

−24% (p < .0001)

0.004% (p = .95) 3.45% (p = .065)

Normalization in all subjects in pooled analysis at 1 yr

Decreased in 11 subjects in pooled analysis at 1 yr

ND

ND

−18.9% (p = .009) normalized in 72%

−38% normalization in 95%

Decreased from BL (p < .0052)

20% (p < .001)

Spleen volume

Decreased from BL (p < .0001) normalization in 100%

−25% (p < .001) normalization in 80%

Liver volume (mean decrease)

Improvement in 9 subjects in pooled analysis at 1 yr

2%

5.6 (p = .009)

ND

ND

FVC % predicted

Improved in shoulder 5 subjects in pooled analysis

ND

ND

Improved in shoulder

Improved in elbow

No change

No consistent improvements

17.4o improvement 46% of subjects improved

Change in affected subjects

Sustained increase

Improved in shoulder, elbow, knee

Joint ROM

(continued on next page)

Improved CHAQ/HAQ

3SMC improved 9.7 steps/min at Week 72 uKS reduction at Week 72: 32.2% 3SMC did not show significant improvement uKS showed consistent reduction

Improved CHAQ/HAQ

–

–

–

–

–

Increased growth rate Improved NYHA score (1–2 classes) Improved breathing/ sleep Increased growth rate from BL Improved NYHA score sustained Improved breathing/ sleep sustained Improved sleep apnea in affected subjects No change in CHAQ/ HAQ Improved sleep apnea in affected subjects Improved CHAQ/HAQ

Other

E. Kakkis and D. Marsden

Molecular Genetics and Metabolism 130 (2020) 7–15

1 mg/kg QW for 24 wks

1 mg/kg QW for 97–260 wks

RDBPC (n = 39)

Open-label (n = 56)

Phase 3 [6]

Phase 1/2, Phase 2, Phase 3 Extensions [49]

RDBPC (n = 12)

Phase 3 [7]

4 mg/kg

4 mg/kg QOW

Mean increase of 20.8 m

1 subject worsened, 1 had no change, 10 was not assessable

ND

NA

ND

130 m 255 ma 117 ma

−79% −72% −71%

No improvement MVV1 improved

The 1 subject assessed showed 13.4% increase from BL at Week 30

92 ma (p = .025)

−75% (p < .001)

Improvement of > 10% in half of the subjects

FVC % predicted

ND

211 ma (p = .002)

−76%

Rapid and sustained dosedependent reduction 58.17% reduction from BL at 240 wks 64.8% reduction at 24 wks

Walk test

uGAG %

No significant change in spleen volume ND

ND

ND

NA

ND

Decrease in affected subjects

Spleen volume

Consistent reduction in liver volume

Significant reduction in enlarged liver size

NA

ND

Decreased in affected subjects normalization in 80%

Liver volume (mean decrease)

11/12 subjects had no change; 1 subject worsened

The 1 patient with limited shoulder ROM resolved and maintained normal range ND

NA

No improvement

ND

Joint ROM

Chondroitin sulfate decrease of 70.6%

ND

ND

Improvement in Timed Up and Go Improved pain and stiffness Improved 3MSC Improvement of 5.7 stairs/min in 3MSC (p = .053) Further improvement in 3MSC

Other

3MSC, 3-Minute Stair Climb; ANCOVA, analysis of covariance; BL, baseline; CHAQ/HAQ, Childhood Health Assessment Questionnaire/Health Assessment Questionnaire; FVC, forced vital capacity; MPS, mucopolysaccharidoses; MVV, maximum voluntary ventilation; NA, not applicable; ND, not done; NYHA, New York Heart Association; QOW, once every other week; QW, once weekly; RDBPC, randomized, double-blind, placebocontrolled study; ROM, range of motion; uGAG, urinary glycosaminoglycan; uKS, urinary keratan sulfate. a 12-Minute Walk Test.

Interventional ( n = 7)

Phase 2 [51]

Mucopolysaccharidosis disease: MPS VII Enzyme replacement therapy: vestronidase alfa Phase 1/2 [50] Forced titration 1–4 mg/kg study (n = 3) titration QOW

1 mg/kg QW for 48 wks

Open-label (n = 10)

Phase 2 [48]

Dose

Design

Development phase

Table 1 (continued)

E. Kakkis and D. Marsden

Molecular Genetics and Metabolism 130 (2020) 7–15

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When evaluating uGAG in subjects with MPS, the clinical status of each individual is relevant in understanding the relationship between the benefit in uGAG reduction and the clinical results. Treatment with ERT from birth before changes are irreversible can prevent many manifestations of MPS disease, as seen in animal models and in published human cases. The overall summary of these data show that uGAG reductions above a threshold of more than 50% are associated with clinically meaningful changes in clinical parameters in the four MPS ERT programs in which uGAG levels were analyzed. However, it is not realistic to expect a linear proportional change in clinical endpoints corresponding to the change in uGAG. Diagnosis is rarely made prior to onset of clinical symptomatology; therefore, substantial reduction in uGAG may have a variable outcome in clinical assessments primarily due to these confounding features. For these reasons, the extent of uGAG reduction may inform treatment decisions but may not be the sole basis for ERT dose selection or modification. Major changes in uGAG levels should be considered as an indicator of biologic activity and might help in assessing the optimal care for patients, or in comparison of alternative dosing regimens or novel ERT products. The processes underlying MPS diseases are well understood and data from studies of ERTs have established that uGAG is a direct pathophysiological marker of the MPS disease process and a reasonable predictor of treatment effect in MPS diseases. Given the strength of existing evidence, uGAG should be considered a relevant biomarker associated with clinical outcomes in MPS diseases. As a biomarker in clinical trials, uGAG may also provide strong supportive evidence for regulatory filings and facilitate development of next-generation, improved treatments or biosimilars, which can be very difficult to do based on clinical endpoints alone. The growing body of evidence supporting the use of uGAG levels as a biomarker of treatment efficacy has the potential to impact long-term outcomes for patients with MPS disorders. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ymgme.2020.02.006.

uGAG reductions of more than 50% with clinical benefit in patients with MPS VII. 2. Clinical outcomes and uGAG reduction in MPS I, II, VI, and VII To establish a predictive relationship between uGAG and clinical effect, a review of the data from clinical studies with ERT in MPS I, MPS II, MPS VI, and MPS VII was performed. All of these studies assessed uGAG in addition to clinical endpoints (Table 1). Data from these Phase 1/2 and 3 controlled studies provide substantial evidence that uGAG is predictive of treatment effect of ERT and can establish the clinical benefit for ERT in MPS diseases. In general, these studies have shown the following: 1. uGAG excretion declines rapidly within a few weeks after dose initiation and reaches relatively stable levels in advance of improvements in clinical efficacy measures in the randomized controlled studies. This pattern is consistent with the mechanism of action, whereby reduction in uGAG reflects clearance in tissue GAG storage that eventually results in improved organ and body system function, translating into clinical benefit. 2. The reduction in uGAG excretion exceeds approximately 50% from baseline in all controlled clinical studies, and this level of reduction is associated with clinically meaningful changes in outcome parameters. 3. uGAG excretion is sensitive and can distinguish between clinically effective and less clinically effective treatment in dose-ranging studies performed in patients with MPS diseases. 4. uGAG excretion is dynamic and responsive to changes in therapy, including missed ERT doses and treatment discontinuation. Overall, these collective data are a strong indicator of the predictive value of uGAG in these MPS diseases. 3. Conclusions

Acknowledgments Taken together, the preclinical and clinical evidence for uGAG reduction as a reasonable estimation of likely clinical benefit following initiation of ERT is compelling though certainly not perfect. Animal models of MPS I, II, VI, and VII demonstrate that change in uGAG excretion is a reasonably sensitive measure with a large dynamic range that correlates with and predicts GAG levels and pathology in multiple tissues. Consistent correlations have been observed between uGAG levels and GAG clearance in the kidney and other tissues, indicating that uGAG is a robust and sensitive indicator of effect not only in less perfused organs, but also in those that are highly responsive to ERT in organs, such as the liver and spleen. In this regard, uGAG levels are a more rigorous measure of treatment effect than measures such as liver storage/size because uGAG levels reflect a wide range of tissue effects, which is important when predicting or assessing treatment effectiveness. Levels of uGAG excretion have been evaluated as the primary biological measure for clinical activity in all MPS ERT programs to date and have demonstrated important relationships with tissue pathology and clinical outcomes. Although uGAG levels may not precisely predict quantitative changes in particular clinical endpoints, and there are insufficient published data to demonstrate correlation of subject-specific uGAG reduction with clinical outcome, the data clearly show that adequate reductions in uGAG more than 50% correlated with important clinical changes in Phase 3 ERT trials. The predictive value is likely impacted by numerous clinical factors such as degree of enzyme deficiency, subject age, disease state, and immunogenicity status. Regardless, only subjects with large reductions in uGAG show improvement consistently and, if ERT is initiated early in life, these changes in uGAG excretion may predict long-term, near-normal outcomes in some subjects.

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