Transcranial pulsed ultrasound facilitates brain uptake of laronidase in enzyme replacement therapy for Mucopolysaccharidosis type I disease

The animal work was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (USA). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Cheng Hsin General Hospital. The C57BL/6 mice (B6 mice) were obtained from the National Laboratory Animal Center; the MPS I mice (B6.129S4-Idua^tm1.1Kmke) with Idua gene knock-in mutation on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were bred and maintained by the National Laboratory Animal Center. At the time of treatment, the C57BL/6 mice had a body weight of 19–24 g, and the MPS I mice had a body weight of 25–31 g.

The pulsed ultrasound was generated by a 1-MHz single element transducer (A392S, Panametrics, Waltham, MA, USA) with a diameter 38 mm and a radius of curvature 63.5 mm. A cone filled with distilled and degassed water was mounted onto the transducer, and the cone tip was capped by a polyurethane membrane. The transducer with cone water was fixed to a stereotactic apparatus (David Kopf Instruments, Tujunga, CA, USA), allowing movement of the transducer to different sonication targets, and was driven by a functional generator (33220A, Agilent Technologies, CO, USA) and a power amplifier (75A250A, Amplifier Research, Souderton, PA, USA). The mouse was in a prone position with its head beneath the cone tip. Ultrasound transmission gel was used to ensure good contact between the head and cone tip (Fig. 1).

The plasma pharmacokinetics of laronidase was investigated in MPS I mice injected with 2.9 mg/kg (5 times the clinical dose used in enzyme replacement therapy) intravenously. Blood samples were collected for IDUA enzyme activity assay at 0.5, 1, 2, 4, 6, 8 and 24 h after injection to find out the optimal time point for laronidase accumulation. IDUA enzyme activity of the liver was compared to that of the brain under normal circumstances.

To study laronidase delivery, the MPS I mice were divided into 4 groups. Group one (n = 3) received no treatment; group two (n = 4) received tail-vein injection of laronidase 2.9 mg/kg; group three (n = 4) received injection of laronidase 2.9 mg/kg followed by one-spot ultrasound exposure (Fig. 2a), and group four (n = 4) received two injections of laronidase 1.45 mg/kg (total 2.9 mg/kg), followed by two-spot ultrasound exposure (Fig. 2b). The IDUA enzyme activity in brain was compared between MPS I mice and normal B6 mice (n = 4) to assess therapeutic effects. In one-spot ultrasound exposure group, laronidase 2.9 mg/kg plus MBs 150 uL/kg were injected just before ultrasound exposure (acoustic pressure 0.56 MPa, pulse repetition frequency [PRF] 1 Hz, burst length 10 ms and sonication duration 60 s). In two-spot ultrasound exposure group, a total dose of MBs 150 uL/kg plus laronidase 2.9 mg/kg was injected in two half-injection, each half was injected just before each spot ultrasound exposure (acoustic pressure 0.56 MPa, PRF 1 Hz, burst length 10 ms and sonication duration 60 s for each spot). The mice were euthanized 4 h after treatment.

EB was used to simulate the distribution of laronidase in the brain. With the same acoustic parameters used in MPS I mice, the B6 mice were subjected to the BBB opening procedure, and EB 100 mg/kg instead of laronidase was delivered into the brain. The B6 mice were divided into 3 groups: group 1 (n = 5) received EB 100 mg/kg injection only, group 2 (n = 12) received EB 100 mg/kg plus MBs 150 uL/kg injection and the previously described one-spot ultrasound treatment (Fig. 2c), group 3 (n = 6) received two EB 50 mg/kg plus MBs 75 uL/kg injection (total EB 100 mg/kg plus MBs 150 uL/kg) and the previously described two-spot ultrasound treatment (Fig. 2d).

Each mouse was anesthetized by intraperitoneal injection of Zoletil (20 mg/kg; Zoletil®50, Virbac Laboratories, Carros, France) plus Xylazine (5 mg/kg; Rompun™, Bayer, Shawnee Mission, KS, USA) before procedures. For mice receiving intravenous laronidase or EB injection without ultrasound treatment, the brain was perfused transcardially with normal saline 4 h after drug injection, followed by euthanasia, and then the brain was harvested and assayed for EB or laronidase.; for mice receiving intravenous laronidase or EB injection with ultrasound treatment, the mice were fixed to the stereotactic apparatus, the tail vein was catheterized. For one-spot ultrasound exposure, the cone of the ultrasound transducer was put 2 mm left lateral of the bregma (Fig. 3a); for two-spot ultrasound exposure, the cone of the ultrasound transducer was put first 1 mm posterior and then 1 mm anterior to the one-spot location (2 mm left lateral of the bregma; Fig. 3b). With the mouse fixed to the stereotactic apparatus and the cone of the ultrasound transducer in place, a solution of MBs (SonoVue®, Bracco, Amsterdam, The Netherlands) mixed with EB (Sigma-Aldrich, St. Louis, MO, USA) or MBs mixed with laronidase (Aldurazyme®, Biomarin Pharmaceutical, San Rafael, CA, USA) was injected through the tail vein and followed immediately by ultrasound exposure. Four hours after treatment, the mouse brain was perfused transcardially with normal saline to wash out the content from the cerebral vasculature, and then the brain was harvested, sliced, and assayed for EB or laronidase.

The mouse brain samples were weighed, placed in trichloroacetic acid solution, homogenized, and centrifuged. The extracted EB was diluted with 95% ethanol in a ratio of 1:3, then put into multiwell-plate-reading fluorometer with excitation wavelength set to 620 nm (X-Zell Biotec, Bangkok, Thailand) and absorption wavelength set to 680 nm. The fluorescence was converted to concentration using a standard curve of known EB concentrations.

IDUA enzyme activity was measured by a fluorometric assay. Mouse brain samples were homogenized, lysed with a lysis buffer, and centrifuged. The substrate of the IDUA, 4-methylumbelliferyl alpha-l-iduronide (4MU-I, Toronto Research Chemicals, Toronto, Ontario, Canada, #M334701), was diluted with sodium formate buffer (50 mM, pH 2.8), then 25 μL aliquots of 4MU-I (200 μM) were mixed with 25 μL aliquots of tissue homogenates. The mixture was incubated at 37 °C for 20 h, and then 200 μL glycine carbonate buffer (pH 10.5) was added to end the reaction. The intensity of emitted fluorescence resulted from enzyme-substrate reaction was measured with excitation at 365 nm and emission at 450 nm. A standard curve was made by 4-Methylumbelliferone (4MU, Sigma-Aldrich #M1381). Protein was determined by Pierce BCA protein assay kit (ThermoFisher scientific #23227). IDUA enzyme activity was expressed as units/mg protein * 20 h.

Our analysis of laronidase pharmacokinetics in MPS I mice plasma showed that the plateau of IDUA enzyme activity persisted 4 h after laronidase injection (Fig. 4), suggesting that activity in the brain and liver should be measured 4 h after treatment. Although liver IDUA enzyme activity of MPS I mice was significantly higher in those injected with laronidase 2.9 mg/kg than those without treatment (Fig. 5), brain IDUA enzyme activity was similar between the groups (Fig. 6, group 1 and group 2), indicating that the BBB blocks the entry of laronidase to the brain. For MPS I mice receiving laronidase and ultrasound treatment (Fig. 2a and b), the brain IDUA enzyme activity on the ultrasound-treated side was significantly higher than that of the control group (Fig. 6, group 3 and group 4); the brain IDUA enzyme activity on the treated side was 2.75-fold and 7.81-fold that on the untreated side after one-spot and two-spot ultrasound treatment, respectively. Compared to normal B6 mice, MPS I mice receiving one-spot and two-spot ultrasound had 30.9 and 75.8% of normal brain IDUA enzyme activity on treated side, respectively.

EB was used to simulate the distribution of laronidase in the brain. The EB molecule (961 Da) binds to albumin (69 kDa) [20] in plasma to form an EB-albumin conjugate (8–14 EB molecules binds to one albumin molecule [21]), which is similar in size to laronidase (83 kDa). In B6 mice receiving ultrasound treatment for brain EB delivery (Fig. 2c and d), brain EB delivery induced by one-spot and two-spot ultrasound treatment schemes with the same acoustic parameters used in MPS I mice resulted in a diffuse pattern of EB distribution throughout the acoustic field (Fig. 7a to c). The amount of EB delivered to the brain on the treated side was 2.83-fold and 3.87-fold that on the untreated side after one-spot and two-spot ultrasound exposure, respectively (Fig. 7d).