Treatment of transmissible spongiform encephalopathy by intraventricular drug infusion in animal models

Abstract

The therapeutic efficacy of direct drug infusion into the brain, the target organ of transmissible spongiform encephalopathies, was assessed in transgenic mice intracerebrally infected with 263K scrapie agent. Pentosan polysulfate (PPS) gave the most dramatic prolongation of the incubation period, and amphotericin B had intermediate effects, but antimalarial drugs such as quinacrine gave no significant prolongation. Treatment with the highest dose of PPS at an early or late stage of the infection prolonged the incubation time by 2.4 or 1.7 times that of the control mice, respectively. PPS infusion decreased not only abnormal prion protein deposition but also neurodegenerative changes and infectivity. These alterations were observed within the brain hemisphere fitted with an intraventricular infusion cannula but not within the contralateral hemisphere, even at the terminal disease stage long after the infusion had ended. Therapeutic effects of PPS were also demonstrated in mice infected with either RML agent or Fukuoka-1 agent. However, at doses higher than that providing the maximal effects, intraventricular PPS infusion caused adverse effects such as hematoma formation in the experimental animals. These findings indicate that intraventricular PPS infusion might be useful for the treatment of transmissible spongiform encephalopathies in humans, providing that the therapeutic dosage is carefully evaluated.

FIG. 1.

Efficacy of quinacrine (A), amphotericin B (B), and PPS (C) in intracerebrally infected mice. Intraventricular infusion of a chemical was initiated at an early infection stage (day 10 postinoculation) or at a late stage (day 35) in 263K-infected Tg7 mice and continued for 4 weeks. Each circle or triangle represents an individual mouse. Bars represent the means and standard deviations of the incubation times for each group. *1, P < 0.05 versus the neighboring group and P < 0.01 versus the other groups; *2, P < 0.05 versus the vehicle control; #1, P < 0.05, #2, P < 0.01.

FIG. 2.

Dose response (A) and administration timing (B) in PPS efficacy. Intraventricular infusion at the indicated concentrations (A) or at 110 or 460 μg/kg/day (B) was initiated at day 42 postinoculation (A) or at the indicated times (B) in intracerebrally 263K-infected Tg7 mice. *1, P < 0.01 versus the other groups; *2, P < 0.01 versus the other groups except the neighboring group(s); *3, P < 0.05 versus the control; *4, P < 0.05 versus the neighboring group and P < 0.01 versus the other groups; *5, P < 0.01 versus the control; #, P < 0.01.

FIG. 3.

Histopathology of the brain with or without PPS treatment. (A) Histology of the brain from an intracerebrally 263K-infected, long-surviving mouse treated with PPS at 460 μg/kg/day from day 10 postinoculation. Noncannula side and cannula side represent the sides of pathogen inoculation and intraventricular cannula implantation, respectively. Hematoxylin-eosin staining was used. (B) Immunohistochemical detection of abnormal PrP deposition (PrP) and of neurodegenerative changes by means of glial reaction (GFAP) in the same brain as for panel A (PPS) or in the brain from a nontreated mouse (control). The orientation of the sides of pathogen inoculation and cannula implantation is the same as in panel A. A coronal section sited around one-third of the distance from the interaural line to the bregma line is shown for the control mouse, and either a coronal section sited around one-third of the distance or a coronal section sited around two-thirds of the distance from the interaural line to the bregma line is shown for the PPS-treated mouse. Astrocytic glial reaction is demonstrated by immunohistochemistry for GFAP. (C) Sequential analysis of abnormal PrP deposition in intracerebrally 263K-infected, nontreated mice (control) or mice treated with PPS (infusion from day 10 or 35). Each panel shows a representative finding from three mice sacrificed at a designated time. In PPS-treated mice, the brain tissue examined was obtained from the hemisphere implanted with the intraventricular cannula, which was the hemisphere opposite to that of the inoculation site. PPS was infused at 460 μg/kg/day from day 10 or 35 postinoculation. Each image of the hippocampus or the thalamus (posterior nuclei) is from coronal sections sited around one-third of the distance from the interaural line to the bregma line. Each image of the pons (ventral area) is from a coronal section which is parallel to the interaural line and contains the culmen portion of the cerebellum. Bar, 160 μm.

FIG. 4.

PrPres in the brain with or without PPS treatment. Each lane represents an aliquot corresponding to 2.0 mg of brain tissue homogenate from an individual mouse sacrificed at a designated time. The brain tissue homogenate examined was prepared from the whole hemisphere contralateral to that of the inoculation site in either nontreated mice (control) or PPS-treated mice (infusion from day 10 or 35). In PPS-treated mice, the brain hemisphere examined was the side implanted with the intraventricular cannula. PPS was infused at 460 μg/kg/day from day 10 or 35 postinoculation. Nonspecific signals that were observed without the primary antibody are shown by arrows.

FIG. 5.

Efficacy of PPS in mice infected with RML agent (A) or Fukuoka-1 agent (B). PPS at a dose of 230 μg/kg/day was intraventricularly infused into intracerebrally infected Tga20 mice from day 14 or 49 postinoculation for 4 weeks. *1, P < 0.01 versus the other groups; *2, P < 0.05 versus the control; *3, P < 0.01 versus the control.

FIG. 6.

Representative data from PPS safety assessment. Platelet counts in dogs and rats (A and D, respectively) and coagulation data for dogs (B and C) are shown. Each circle, triangle, square, inverted triangle, or diamond represents an individual animal. The data for two of the six dogs receiving 345 μg/kg/day and for three of the four dogs receiving 460 μg/kg/day were not obtained because they suffered seizures shortly after PPS infusion had been started and were sacrificed or died. However, the data for two dogs, one receiving 345 μg/kg/day and the other receiving 460 μg/kg/day, both of which suffered seizures but recovered, are included.