Application of N-palmitoyl-O-phosphocholineserine for diagnosis and assessment of response to treatment in Niemann-Pick type C disease

Abstract

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder caused by mutations in either the NPC1 or the NPC2 gene. A new class of lipids, N-acyl-O-phosphocholineserines were recently identified as NPC biomarkers. The most abundant species in this class of lipid, N-palmitoyl-O-phosphocholineserine (PPCS), was evaluated for diagnosis of NPC disease and treatment efficacy assessment with 2-hydroxypropyl-β-cyclodextrin (HPβCD) in NPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated to measure PPCS in human plasma and cerebrospinal fluid (CSF). A cutoff of 248 ng/mL in plasma provided a sensitivity of 100.0% and specificity of 96.6% in identifying NPC1 patients from control and NPC1 carrier subjects. PPCS was significantly elevated in CSF from NPC1 patients, and CSF PPCS levels were significantly correlated with NPC neurological disease severity scores. Plasma and CSF PPCS did not change significantly in response to intrathetical (IT) HPβCD treatment. In an intravenous (IV) HPβCD trial, plasma PPCS in all patients was significantly reduced. These results demonstrate that plasma PPCS was able to diagnose NPC1 patients with high sensitivity and specificity, and to evaluate the peripheral treatment efficacy of IV HPβCD treatment.

Keywords: 2-Hydroxypropyl-β-cyclodextrin; Diagnosis; LysoSM-509; N-palmitoyl-O-phosphocholineserine; Niemann-Pick disease type C; Treatment assessment.

Figure 1.

Plasma PPCS in patients affected with lysosomal storage diseases. (A) PPCS in plasma samples from control (n = 130), NPC1 heterozygote (n = 48), and NPC1 affected individuals. (n = 179). Data are presented as mean ± SEM. P < 0.0001 for heterozygotes versus controls and for NPC1 versus controls. (B) ROC curve demonstrates 0.9992 area under the curve for PPCS. A cut-off value of 248 ng/mL yields a sensitivity of 100% and specificity of 96.63% to discriminate NPC1 affected individuals from controls and NPC1 heterozygotes. (C) PPCS in plasma samples from patients with Fabry (n = 12), FH (n = 4), Gaucher (n = 2), Krabbe (n = 2), CTX (n = 2), GM1 (n = 1), GM2 including Tay-Sachs (black, n = 3) and Sandhoff (red, n = 2), and SPG5 (n = 1) diseases. Data are presented as mean ± SEM. (D) PPCS in plasma samples from patients with MPS I (black, n = 2), II (brown, n = 2), IIIA (orange, n = 2), IIIB (red, n = 2), IIIC (green, n = 2), IVA (pink, n = 2), VI (purple, n =2), VII (blue, n =2), Batten (CLN1, red, n = 1; CLN2, pink, n = 1; CNL3, black, n = 20), MLII/III (n = 2), SYNGAP1 (n = 1), CESD (n = 1), Wolman (n = 1), ASMD (n = 11) diseases, and ASMD carriers (n = 3). Data are presented as mean ± SEM. (E) Correlation of plasma PPCS levels with NPC neurological disease severity scores. (F) correlation of plasma PPCS levels with NPC annual severity increment scores. (G) PPCS in the plasma over time collected from the natural history study. (H) Comparison of plasma PPCS levels in male and female NPC1 patients.

Figure 2.

Response of plasma PPCS levels to HPβCD treatment in NPC1 study participants. (A) Variation of plasma PPCS during 72 hours in IT saline and 900 mg HPβCD treatments. (B) NPC1 study participants with reduced plasma PPCS in response to monthly IT HPβCD treatment. (C) NPC1 study participants with increased plasma PPCS in response to monthly IT HPβCD treatment. (D) NPC1 study participants with unchanged plasma PPCS in response to monthly IT HPβCD treatment. (E) Response of plasma PPCS to weekly IV HPβCD treatment.

Figure 3.

PPCS levels in human CSF. (A) PPCS in CSF samples from control (n = 40) and NPC1 affected individuals. (n = 23). Data are presented as mean ± SEM. P < 0.0001 for NPC1 versus controls. (B) Correlation of CSF PPCS levels with NPC neurological disease severity scores. (C) Correlation of CSF PPCS levels with NPC disease annual severity increment scores. (D) Correlation in PPCS levels between CSF and plasma. (E) Response of CSF PPCS to monthly IT HPβCD treatment.

Figure 4.

PPCS in cat serum. (A) Correlation in PPCS levels between cat plasma and serum. (B) Serum PPCS in normal cats. (C) Serum PPCS in NPC1 heterozygous cats. (D) Serum PPCS in untreated NPC1 cats. (E) Serum PPCS in NPC1 cats treated with saline every other week. (F) Serum PPCS in NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 3 weeks. (G) Serum PPCS in NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 16 weeks. (H) Serum PPCS in NPC1 cats treated with IC 120 mg/kg HPβCD and SC 1000 mg/kg HPβCD every other week beginning at 3 weeks.

Figure 5.

PPCS in cat liver and brain tissues. (A) PPCS in liver of normal cats (n = 9, 23–59 weeks), heterozygous cats (n = 5, 24 – 253 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD and SC 1000 mg/kg HPβCD every other week beginning at 3 weeks (n = 7, 65 – 182 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 3 weeks (n = 3, 121 – 145 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 16 weeks (n = 3, 42 – 51 weeks), NPC1 cats treated with IC saline every other week beginning at 3 weeks (n = 3, 21 – 26 weeks), and untreated NPC1 cats (n = 5, 20 – 27 weeks). Data are presented as mean ± SEM. P = 0.0002 for untreated NPC1 cats versus normal cats. (B) PPCS in cat brains of normal cats (n = 10, 23 – 59 weeks), heterozygous cats (n = 5, 24 – 253 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD and SC 1000 mg/kg HPβCD every other week beginning at 3 weeks (n = 7, 65 – 182 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 3 weeks (n = 3, 121 – 145 weeks), NPC1 cats treated with IC 120 mg/kg HPβCD every other week beginning at 16 weeks (n = 3, 42 – 51 weeks), NPC1 cats treated with IC saline every other week beginning at 3 weeks (n = 3, 21 – 26 weeks), and untreated NPC1 cats (n = 6, 20 – 27 weeks). Data are presented as mean ± SEM. P = 0.0001 for untreated NPC1 cats versus normal cats. (C) Correlation in PPCS levels between serum and liver. (D) Correlation in PPCS levels between serum and brain.