Bone turnover and mineral metabolism in adult patients with hypophosphatasia treated with asfotase alfa

Hypophosphatasia (HPP) is a rare, inherited, metabolic disease caused by a tissue nonspecific alkaline phosphatase (TNSALP) deficiency, leading to the extracellular accumulation of TNSALP substrates and further resulting in impaired bone mineralization, skeletal defects, and systemic manifestations of the disease [1‐3]. Adult patients with HPP can experience a high disease burden, including a wide range of dental and orthopedic problems such as fractures, pseudofractures, bone and joint pain, muscle weakness, and fatigue. Consequently, patients with HPP have reduced physical functioning and health-related quality of life [3‐6].

The pathophysiology of HPP involves extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP substrate, which inhibits bone mineralization by blocking hydroxyapatite crystal formation [2]. Asfotase alfa is a human recombinant TNSALP enzyme replacement therapy that is approved for HPP treatment, typically for patients of all ages with pediatric-onset HPP (all adults regardless of onset in Japan) [7]. Prior research has shown that asfotase alfa treatment reduces the accumulation of TNSALP substrates, PPi, and pyridoxal 5′-phosphate (PLP), and promotes bone mineralization [8, 9]. However, assessment of PPi is not available as a routine clinical assay and is currently used in research settings only. Other established indicators of TNSALP activity, such as PLP and urine phosphoethanolamine (PEA) [1, 2, 10], are not implemented for monitoring asfotase alfa treatment in routine practice since clinically available PLP assays do not use an inhibitor of alkaline phosphatase (ALP) activity (e.g., levamisole) to prevent PLP decay in vitro by arresting asfotase alfa activity in serum samples of treated patients [9, 11], and PEA has not been evaluated in that context.

Along with reduced ALP activity and the accumulation of its substrates, poor bone mineralization and bone turnover appear to be hallmarks of HPP. Indeed, previous studies have indicated diminished bone turnover along with reduced serum ALP activity [12, 13], and elevated calcium and/or phosphate levels in patients with HPP may be considered sequelae of poor bone mineralization [14‐16]. While case reports from adult patients with HPP suggest that asfotase alfa has a positive clinical effect on bone health [17, 18], limited data are available on how treatment with asfotase alfa affects mineral metabolism and bone turnover, and how these treatment effects can be biochemically monitored during routine clinical care. Therefore, this study characterized and evaluated parameters of bone turnover and ALPL-associated mineral metabolism in adult patients with pediatric-onset HPP who were treated with asfotase alfa in a routine clinical practice setting.

Baseline patient demographics and treatment characteristics are shown in Table 1. A total of 21 adult patients (n = 16, female) with pediatric-onset HPP, aged 19–78 years (median 50 years), were included in the analysis. All patients had genetically confirmed HPP (Supplemental Table S1) with consistently reduced serum ALP activity and bone manifestation of the disease, including a positive fracture history in 20 patients. Additional bone manifestations of HPP in the study population were bowing of the long bones (n = 9), craniosynostosis (n = 7), scoliosis (n = 5), and kyphosis (n = 3). Although all adult patients had a diagnosis of pediatric-onset HPP, 14 (67%) of 21 patients did not experience a first fracture until adulthood. Almost all patients (20 [95%] of 21 patients) reported further manifestations of HPP, such as pain (n = 20; 95%), muscle weakness that limited daily activities (n = 19; 90%), joint pain (n = 18; 86%), and early loss of primary (n = 17; 81%) and permanent (n = 9; 43%) dentition.

Serum ALP activity, urine PEA/Cr ratio, and GFR levels were assessed at every visit from baseline through 24 months of treatment; results are shown in Fig. 1A–C. As expected, serum ALP activity significantly increased when compared with baseline at every study timepoint following the start of treatment with asfotase alfa. Median (Q1, Q3) urine PEA/Cr ratio levels were increased at baseline (53.2 mmol/mol Cr [37.3, 78.3]) and reduced to near the normal range after treatment initiation with asfotase alfa. Compared with baseline, this decrease was statistically significant at every timepoint assessed (6, 12, 18, and 24 months); however, median urine PEA/Cr ratio levels remained above the upper limit of the normal range throughout the 24-month follow-up period.

Renal function was continuously monitored throughout the study, specifically since some patients had reduced baseline GFR levels equivalent to chronic kidney disease (CKD) grades 2 and 3. Although median GFR levels did not change significantly over the course of the study, individual GFR levels at certain timepoints dipped below 60 mL/min/1.73m²; however, these decreases appeared to be transient, and severe renal compromise was not observed in any patient during the course of treatment.

Median (Q1, Q3) serum levels for markers of mineral metabolism (PTH, calcium, phosphate) and FGF-23 in study participants at baseline and 3, 6, 12, 18, and 24 months of treatment are shown in Fig. 2A–D. Significant increases in PTH were observed at 3 months (41.7 pg/mL [24.9, 51.5]; P = 0.008) and 6 months (38.1 pg/mL [26.6, 46.0]; P = 0.018) of treatment when compared with baseline (27.6 pg/mL [23.6, 36.9]). There were no significant changes in median serum levels of calcium and phosphate at any study timepoint when compared with baseline. Median serum PTH, calcium, and phosphate levels stayed within normal limits throughout the analysis. Similarly, median (Q1, Q3) serum levels for FGF-23, an essential regulator of serum phosphate homeostasis, did not significantly change from baseline (84 RU/mL [54, 103]) through 24 months of treatment with asfotase alfa and remained within normal limits. Outliers ranging between 1.5- and 3.0-fold above the upper limit of the normal range stem from one patient with CKD grade 3 and GFR levels consistently between 45 and 60 mL/min/1.73m².

Median (Q1, Q3) serum levels (osteocalcin, P1NP, TRAP5b, and NTx) for bone turnover markers are shown in Fig. 3A–D. Median serum osteocalcin levels increased significantly at 3 months (18.8 ng/mL [10.9, 23.4]; P = 0.001) and 6 months (16.3 ng/mL [11.5, 22.9]; P = 0.001) when compared with baseline (10.4 ng/mL [8.5, 14.4]), and then returned to near baseline levels after 12 months (12.4 ng/mL [10.4, 19.4]) and remained within normal limits through 24 months (13.7 ng/mL [9.3, 18.5]) of treatment with asfotase alfa. Similarly, median serum P1NP levels increased significantly at 3 months (96.7 μg/L [72.8, 127.0]; P = 0.002) and 6 months (70.1 μg/L [56.9, 98.2]; P = 0.035) compared with baseline (56.0 μg/L [46.0, 70.0]) and returned to near baseline levels at 12 months (56.8 μg/L [49.7, 72.9]), remaining within normal limits through 24 months (54.6 μg/L [43.6, 66.7]) of treatment.

In parallel, TRAP5b levels increased significantly at 3 months (4.2 U/L [2.8, 5.2]; P = 0.009) when compared with baseline (3.1 U/L [2.6, 4.1]) and returned towards baseline levels at 12 months (2.9 U/L [2.3, 4.7]), remaining within normal limits at all timepoints from baseline through 24 months (2.7 U/L [1.6,3.9]) of treatment. Compared with baseline NTx levels (11.8 nM BCE/L [10.0, 17.2]), there was a numerical increase in NTx levels at 3 months (13.2 nM BCE/L [9.9, 17.0]; P = 0.435) and 6 months (13.3 nM BCE/L [11.1, 16.0]; P = 0.528) that did not reach statistical significance. NTx levels decreased thereafter, with the decline being statistically significant at 24 months (9.5 nM BCE/L [8.2, 10.8]; P = 0.022) when compared with baseline. NTx levels remained within the normal range at all timepoints.

Mean lumbar spine T scores (Fig. 4) were in the upper range of normal at baseline and increased when compared with baseline after 6 and 12 months of treatment with asfotase alfa (P < 0.05). Assessments of DXA at the femoral region were valid for only a few patients, with the majority of them having metal implants, making statistical evaluations not feasible due to the small sample size.

During treatment with asfotase alfa, four bone fractures and three pseudofractures occurred in four patients with a history of fractures. One patient experienced metatarsal pseudofractures of two bones and two traumatic avulsion fractures at the thumb following injury of the hand; one patient experienced an insufficiency fracture at the medial malleolus 3 months after treatment initiation; and one patient had a solitary metatarsal pseudofracture detected. In one patient with a preexisting unstable femoral implant and progressively thinned femoral cortex, a femoral fracture occurred when the patient became more active after starting treatment.

Results of this study show significant changes in bone turnover and mineral metabolism markers in adult patients with pediatric-onset HPP who were treated with asfotase alfa. As expected, serum ALP activity levels increased substantially following treatment with asfotase alfa. Though serum ALP activity may be useful in assessing treatment adherence, we cannot draw any conclusions concerning therapeutic ALP levels and treatment efficacy. In that regard, previous studies have focused on measuring PPi and PLP substrate levels [9, 11, 16, 21, 22].

Unfortunately, monitoring these substrates in a routine clinical setting is impractical because degradation of PPi and PLP after drawing blood must be prevented by the addition of a standardized concentration of an ALP inhibitor, such as levamisole, to test tubes. In addition, samples for PPi would have to be kept on ice and filtered immediately for use in a clinical setting [23, 24], but neither levamisole-treated test tubes nor clinically approved PPi assays are commercially available, yet.

Accordingly, as part of this observational study, we evaluated whether assessing levels of the glycophospholipid, PEA, normalized to Cr in urine (urine PEA/Cr ratio), was feasible and responsive to treatment with asfotase alfa in adults with HPP. In line with that hypothesis, urine PEA/Cr ratio levels were significantly reduced at all study timepoints (6, 12, 18, and 24 months) following treatment initiation when compared to baseline. However, median urine PEA/Cr ratio levels did not reach the normal range, which is similar to the findings in a previous report [25]; instead, median urine PEA/Cr ratio levels remained slightly above the upper limit of the normal range. Unfortunately, normal range values for urine PEA/Cr ratio levels are generally poorly defined with ranges varying among different laboratories. Therefore, drawing conclusions from specific target values is difficult, specifically since there is no established target range for urine PEA/Cr ratio levels in adults with HPP. Accordingly, we cannot confirm whether the degree of reduction or absolute urine PEA/Cr ratio levels attained following the initiation of asfotase alfa are meaningful with regard to treatment response or efficacy. Even though PEA is considered a natural substrate of ALP [26], the metabolic pathway eventually leading to PEA accumulation in HPP is not fully understood [27, 28]. This is because large amounts of PEA are supposed to be derived from the phosphatidylinositol glycan moiety that couples proteins such as ALP to the cell surface, and the possibility that the majority of PEA is derived from the liver rather than bone [29]. Reflecting on these limitations, it appears premature to recommend urine PEA/Cr ratio levels as a pivotal marker for treatment efficacy or dose adjustments. Still, if additional studies can support the significance of this marker for monitoring treatment, this may become expedient in the future.

In this study, baseline serum levels for all bone and mineral metabolism markers were near normal. However, along with reduced serum ALP activity, baseline levels for PTH, osteocalcin, and NTx were detected toward the lower end of the normal range. These findings are consistent with previous reports indicating that there are reduced mineral turnover, diminished bone metabolism, and, consequently, compromised uptake of circulating minerals to the bone prior to treatment in patients with HPP [12, 13, 16]. Correspondingly, calcium and specifically phosphate levels tended to be at the higher end of the normal range. Importantly, none of the patients in this study was exposed to osteoporosis drugs for at least the last 5 years before initiation of ERT. Accordingly, a relevant impact of such drugs or their withdrawal on bone turnover markers in this study is highly unlikely.

Treatment initiation in this cohort elicited considerable alterations in mineral metabolism and bone turnover, likely reflecting corresponding changes in bone remodeling. Compared with baseline, there were significant increases in PTH at 3 and 6 months of treatment, while serum calcium, phosphate, and FGF-23 levels did not change significantly across 24 months of treatment with asfotase alfa. Building on the understanding that asfotase alfa degrades mineralization inhibitors such as PPi and potentially phospho-osteopontin, these changes may reflect a compensatory mechanism to address a transiently increased need for calcium, while on the other hand, the requirement for additional phosphate can be compensated by phosphate released from previously accumulated ALP substrates.

In contrast to other bone-targeted treatment strategies such as bisphosphonates, PTH/parathyroid hormone-related protein analogues, or antibodies against receptor activator of nuclear factor kappa-Β ligand (RANKL) or sclerostin, enzyme replacement therapy with asfotase alfa is not known to directly and specifically interfere with cell differentiation, proliferation, activity, or apoptosis. However, even though asfotase alfa is essentially just reenabling and catalyzing a previously insufficient chemical process, our data clearly show that beyond merely facilitating degradation of phosphoric compounds, treatment with asfotase alfa eventually has an impact on osteoblast and osteoclast-mediated bone remodeling. Considering that bone turnover and remodeling is only feasible on mineralized bone surfaces, it appears reasonable to assume that mineralization of previously unmineralized osteoid (i.e., healing of osteomalacia following asfotase alfa treatment) could enable remodeling activity particularly on previously inaccessible bone surfaces. Specifically, there was a prolonged increase in TRAP5b levels, likely reflecting increased differentiation and formation of osteoclasts. Similarly, osteocalcin and P1NP were significantly elevated during the first 6 months of treatment with an increase in osteocalcin being suggestive of increased osteoblast activity and bone mineralization [30, 31], whereas elevated P1NP indicates increased type 1 collagen synthesis and deposition. In parallel, actual resorptive activity determined by NTx exhibited a nonsignificant trend to be transiently increased and eventually decreased, suggesting a progressively stable situation during the course of treatment without further need for enhanced remodeling.

In line with previous reports, lumbar spine BMD scores in this cohort of severely affected HPP patients were higher than femoral BMD scores and above average for the normal population [32, 33]. A potential explanation for this finding was the accumulation of osteoid and inappropriately mineralized, mechanically inferior bone tissue, specifically in axially loaded vertebrae as a consequence of underlying osteomalacia with compromised bone quality and reduced turnover. In addition, erroneous calcification of ligamentous structures may contribute to high lumbar spine BMD scores in patients with HPP. Following treatment initiation with reduction of mineralization inhibitors such as PPi, progressive mineralization of both these may have contributed to the observed further increase in lumbar spine BMD.

Because ALP activity reduces the level of mineralization inhibitors and elevated ALP activity is associated with vascular calcification in CKD, concerns regarding an increased risk of vascular calcification during treatment with asfotase alfa have been raised based on animal models of excessive ALP activity [34, 35]; however, no such findings have been documented in humans [36]. Notably, lateral assessments regularly done in addition to our BMD assessments did not reveal any signs of aortal calcification or increased mineral deposition within the longitudinal ligament. Accordingly, the increased BMD scores following initiation of asfotase alfa are most likely associated with bone healing, bone remodeling, and enhanced bone mineralization of preexisting mineralized tissue. Indeed, increases in lumbar spine BMD scores have previously been reported in children on enzyme replacement therapy, and in this setting, improvements in Rickets Severity Score correlated significantly with increased lumbar spine BMD_htZ scores [37].

Regarding fracture incidence, our data provide a first impression on the incidence of fractures following the initiation of enzyme replacement therapy in patients with HPP. However, of the four fractures and three pseudofractures documented, only three metatarsal fractures in two patients and one fracture of the medial malleolar in another patient shortly after treatment initiation can possibly be associated with HPP-related bone quality. Thus, it is premature to draw conclusions related to fracture incidence following treatment initiation.

The main limitations of our study are its observational nature and the limited sample size, which restricts generalizability of our findings. Regardless, considering the rarity of adult HPP patients with severe pediatric-onset HPP and overt bone manifestations, results presented herein reflect the largest available cohort with consistent outcome data in that regard.

Taken together, the observed alterations in mineral metabolism and bone formation markers, together with changes in BMD, further support the understanding that asfotase alfa facilitates bone mineralization, while also providing some insight that treatment with asfotase alfa may elicit bone remodeling and potentially improve bone structure in adults with pediatric-onset HPP.

Results of this study indicate that asfotase alfa treatment is associated with significant changes in biochemical markers of bone and mineral metabolism in adult patients with pediatric-onset HPP. These changes suggest that treatment-mediated mineralization may enable bone remodeling and bone turnover on previously unmineralized and thus inaccessible bone surfaces. In addition, our findings suggest that the urine PEA/Cr ratio should be evaluated further as a potential biochemical marker for monitoring asfotase alfa treatment.