An initial animal proof-of-concept study for central administration of clozapine to schizophrenia patients

Abstract

While clozapine is the acknowledged superior pharmacotherapeutic for the treatment of schizophrenia, the side effect profile, which includes potentially fatal complications, limits its usefulness. Central administration of clozapine directly into the brain could circumvent many of the side effect issues due to the dramatic reduction in dose and the limitation of the drug primarily to the CNS. The present study demonstrates that clozapine can be formulated as a stable solution at physiological pH, which does not have in vitro neurotoxic effects at concentrations which may be effective at treating symptoms. Acute central administration improved auditory gating deficits in a mouse model of schizophrenia-like deficits. Assessment of behavioral alterations in rats receiving chronic central infusions of clozapine via osmotic minipump was performed with the open field and elevated plus mazes. Neither paradigm revealed any detrimental effects of the infusion. While these data represent only an initial investigation, they none-the-less suggest that central administration of clozapine may be a viable alternate therapeutic approach for schizophrenia patients which may be effective in symptom reduction without causing behavioral or neurotoxic effects.

Figure 1

Clozapine (1 mg/ml) was solubilized under acidic conditions, and the solution pH was titrated up toward physiological pH. In the absence of HPBCD, clozapine precipitation at approximately pH 6.5 is indicated by a dramatic increase in light scattering. Formulation of clozapine at increasing HPBCD-to-clozapine mole ratios maintains solubility at progressively higher pH, and precipitation is completely eliminated at mole ratios of 3:1 and 4:1. The ability of HPBCD to maintain clozapine solubility, even under highly alkaline conditions (pH ∼12), is an indicator of how effectively the drug is solubilized under these conditions.

Figure 2

A. This graph shows the cell viability at 24 hours at dilutions of HPBCD in culture media from 0.002% to 0.1%. There was no significant reduction in neuronal viability with the HPBCD solutions used to solubilize clozapine. B. When clozapine was formulated with HPBCD and added to the cells, significant toxicity was observed at final clozapine concentrations of 10 μg/ml (30 μM) or higher. This toxicity is similar to that reported to occur to human neutrophils and monocytes (Gardner et al. 1998).

Figure 3

The 0.1 μg clozapine in 1.0 μl injection of clozapine into the anterior lateral ventricle of male DBA/2 mice did not significantly alter any of the auditory gating variables tested. Four baseline records (b) were obtained prior to ICV administration of the clozapine (at the arrow) and records were obtained at 5 minute intervals for 90 minutes after injection. Data are mean ± SEM, n=8.

Figure 4

The 0.5 μg clozapine in 1.0 μl injection of clozapine into the anterior lateral ventricle of male DBA/2 mice caused a significant reduction in test amplitude which produced a significant reduction in TC ratio. While in increase in conditioning amplitude can be seen in the graph, it failed to attain significance. Five baseline records (b) were obtained prior to ICV injection of clozapine (at the arrow) and records were taken at 5 minute intervals for 95 minutes after injection. Data are mean ± SEM; n=8; * p <0.05, ** p <0.01, Fishers' LSD.

Figure 5

The 1.0 μg clozapine in 1.0 μl injection of clozapine into the anterior lateral ventricle of male DBA/2 mice produced significance increases in conditioning amplitude and decreases in test amplitude which combined to produce a sustained decrease in TC ratio. Five baseline records (b) were obtained prior to ICV injection of clozapine (at the arrow) and records were taken at 5 minute intervals for 95 minutes after injection. Data are mean ± SEM; n=8; * p <0.05, ** p <0.01, Fishers' LSD.

Figure 6

Male Sprague-Dawley rats were implanted with an osmotic minipump delivering either 10 or 17 mg/ml clozapine (0.25 μl/hr) directly into one anterior lateral ventricle. A. In open field testing, the only significant change observed was an increase in distance traveled over the 5 minutes of testing, in the outer region of the open field maze suggesting that the clozapine did not produce any sedation in these animals. B. When tested in the elevated plus maze, the rats only showed significant changes in the amount of time spent in the open arms of the maze, suggesting some anxiolytic effects of the centrally administered clozapine. Together these data demonstrate that chronic central administration of clozapine did not produce any detrimental behavioral effects in these rats. Data are mean ± SEM; n=4 per group; * p<0.05, Fisher's LSD.