Clearance of Hepatic Sphingomyelin by Olipudase Alfa Is Associated With Improvement in Lipid Profiles in Acid Sphingomyelinase Deficiency

Abstract

Acid sphingomyelinase deficiency (ASMD; Niemann-Pick disease type A and B) is a lysosomal storage disorder characterized by abnormal intracellular sphingomyelin (SM) accumulation. Prominent liver involvement results in hepatomegaly, fibrosis/cirrhosis, abnormal liver chemistries, and a proatherogenic lipid profile. Olipudase alfa (recombinant human ASM) is in clinical development as an investigational enzyme replacement therapy for the non-neurological manifestations of ASMD. In a phase 1b study conducted to evaluate the safety and tolerability of within-patient dose escalation with olipudase alfa, measurement of SM levels in liver biopsies was used as a pharmacodynamic biomarker of substrate burden. Five adult patients with non neuronopathic ASMD received escalating doses of olipudase alfa every 2 weeks for 26 weeks. Liver biopsies obtained at baseline and 26 weeks after treatment were evaluated for SM storage by histomorphometric analysis, biochemistry, and electron microscopy. Biopsies were also assessed for inflammation and fibrosis, and for the association of SM levels with liver volume, liver function tests, and lipid profiles. At baseline, SM storage present in Kupffer cells and hepatocytes ranged from 9.8% to 53.8% of the microscopic field. After 26 weeks of treatment, statistically significant reductions in SM (P<0.0001) measured by morphometry were seen in 4 patients with evaluable liver biopsies. The 26-week biopsy of the fifth patient was insufficient for morphometric quantitation. Posttreatment SM levels ranged from 1.2% to 9.5% of the microscopic field, corresponding to an 84% to 92% relative reduction from baseline. Improvements in liver volume, liver function tests, and lipid profiles were also observed. This study illustrates the utility of SM assessment by liver biopsy as a pharmacodynamic biomarker of disease burden in these patients.

Trial registration: ClinicalTrials.gov NCT01722526.

FIGURE 1

MetaMorph quantification of SM in liver biopsies before and after olipudase alfa treatment. This staining and quantification method identifies and measures only abnormal SM accumulation, which is part of the disease process. The normal value in a non-ASMD liver is 0. The “n” for number of blocks analyzed by MetaMorph at baseline and week 26, respectively, for each patient are as follows: Patient 1, n=2, n=0; patient 2, n=4, n=8; patient 3, n=8, n=9; patient 4, n=5, n=6; patient 5, n=7, n=7. *P<0.0001.

FIGURE 2

SM was cleared in Kupffer cells (K) and markedly reduced in hepatocytes (H). A modified toluidene blue stain highlights pretreatment (A) and posttreatment (B) SM in dark purple. Comparable identification of SM is also achieved with lysenin affinity staining, which highlights pretreatment (C) and posttreatment (D) SM in red (patient 2, high-resolution light microscopy, epon semithin sections, 600x).

FIGURE 3

Patterns of hepatic SM accumulation and clearance. The top 3 panels illustrate in cartoon format the 3 primary stages of SM accumulation observed in the patient biopsies. Acinar zones are marked in yellow. The bottom 2 panels show the pattern of SM clearance in a patient biopsy with moderate SM levels (stage 2) at baseline. Pretreatment and posttreatment biopsy images are shown for patient 5. At baseline, substrate accumulation was heaviest around central veins (CV), particularly acinar zones 3 and 2. After 26 weeks of olipudase alfa, there was noticeable clearance of SM in zones 1 and 2, with reduction in zone 3 (Epon semithin sections, lysenin affinity stain). PT indicates portal triad, 20x.

FIGURE 4

Electron microscopy and high-resolution light microscopy examination of SM accumulation and clearance from Kupffer cells. A, The characteristic “fingerprint” whorls of SM present in Kupffer cells at baseline in patient 3. Electron dense lipofuscin (L) is also present. Insets in A: Kupffer cell SM is dark purple in modified toluidene blue sections (left) and red in lysenin affinity sections (right). B, The clearance of SM, with lipofuscin remaining (L). Insets in B: This residual lipofuscin appears light blue in modified toluidene blue sections (left) and blue-green in lysenin affinity sections (right). Electron microscopy scale bars=1 μm.

FIGURE 5

Electron microscopy examination of SM accumulation and clearance from hepatocytes. A, The presence of large SM masses mixed with lipofuscin (L) within hepatocytes at baseline in patient 5. Smaller whorls of SM are also present (black arrows). B, The reduction of SM after 26 weeks of olipudase alfa treatment. Black arrows indicate residual SM. Electron microscopy scale bars=1 μm.