In vitro and in vivo demonstration of risperidone implants in mice

Abstract

Background: Non-adherence with medication is a critical limitation in current long-term treatment of schizophrenia and a primary factor in poor quality-of-life outcomes. However, few treatments have addressed this shortcoming using an implantable drug delivery approach. The goal of this study was to provide in vitro and in vivo proof of concept for a long-term implantable risperidone delivery system in mice.

Methods: Implantable formulations of risperidone were created using the biodegradable polymer Poly Lactic co Glycolic Acid (PLGA) combined with various drug loads. Implant bioactivity was tested using in vitro release and stability studies, as well as in vivo pharmacokinetic and behavioral studies in mice.

Results: The pattern of risperidone release is influenced by various parameters, including polymer composition and drug load. In vitro measures demonstrate that risperidone is stable in implants under physiological conditions. Behavioral measures demonstrate the bioactivity of risperidone implants delivering 3 mg/kg/day in mice, while pharmacokinetic analyses indicate that reversibility is maintained throughout the delivery interval.

Conclusions: The current report suggests that implantable formulations are a viable approach to providing long-term delivery of antipsychotic medications based on in vivo animal studies and pharmacokinetics. Implantable medications demonstrated here can last two months or longer while maintaining coherence and removability past full release, suggesting a potential paradigm shift in the long-term treatment of schizophrenia.

Figure 1

Insertion and removal of a 1 cm rod-shaped risperidone implant is shown in mouse. A) A 1 cm risperidone implant (inset) and trochar (12 gauge) are shown prior to implantation. B) The mouse was anaesthetized with isoflurane and the skin was shaved prior to cleaning the area with betadine and alcohol. The 1 cm length implant was then inserted through a 3 mm hole using a trochar (similar to a 12 gauge needle). A comparable procedure in humans would utilize a local analgesic, such as lidocaine, to numb the skin prior to insertion. C) The implant site is shown after closing with a single stitch. Note that the implant is visible under the skin (white arrow) and is about the size of a grain of rice. Similar procedures in humans could be closed with steristrips. D) The mouse is shown 10 minutes later in its home cage with no signs of distress. The implant is seen over the dorsal surface (white arrow). E) A mouse is shown at 2 weeks after implantation. The implant site is completely healed with no signs of distress or adverse events noted. The implant remains palpable and visible (white arrow). F) Mice had implants (white arrow) removed 14, 27, 56 and 83 days after implantation to assess reversibility of the procedure. This panel shows an example of a mouse just prior to implant retrieval. The mouse in the picture is anesthetized with isoflurane. G) Implants were easily removed at all time points without adhesions or local scarring. A small incision was made with a scalpel and the implant was identified under the skin prior to retrieval with forceps. Note, that the retrieval procedure would only be necessary in humans if there was a need to end treatment prematurely. If left in place, these implants are fully biodegradable and would not require retrieval. Also, retrieval in a human would require local analgesia with lidocaine or other similar agent. H) A mouse shown back in its home cage 10 minutes after implant removal with no signs of distress. An example of a removed implant is shown in the inset. Note, that the implants retained cohesion, fostering removal throughout the study interval. Mice in these groups were then sacrificed and serum risperidone levels obtained.

Figure 2

Risperidone implants remain stable after ethylene oxide (EO) sterilization and after incubation in a low pH environment. A) The patterns of release from four EO sterilized and four surface sterilized implants are shown. The mean ± SEM for each group is shown at each time point. No significant difference is apparent over six weeks of testing. B) The stability of risperidone in six low pH environments is shown over 180 days. Risperidone is stable at both a neutral pH and low pH as might occur within the local microenvironment of the implant.

Figure 3

In vitro risperidone release varies with polymer composition, but not with geometry over the range tested. A) Cumulative in vitro risperidone release from implants containing 50:50, 65:35, or 75:25 PLGA, 20 wt % drug load. Data are expressed as cumulative % total release over time. Each point represents the mean ± SEM of 3 implants. Note that implants reach full release at about 40, 80, and 120 days respectively with 20 wt % initial drug load. B) The release from three implants with different surface area to volume ratios (1.8, 2.8, 6.2 cm^-1), tested using 30 wt % drug load, did not significantly differ over seven weeks of testing (p = 0.90).

Figure 4

In vitro risperidone release varies with drug load. In vitro cumulative risperidone release from implants containing 85:15 PLGA with 10, 20, 30, 40, 50, or 60 wt % drug load by weight are displayed. A) Cumulative mass of risperidone in vitro is shown for each set of three implants (mean ± SEM). B) The pattern for the 40 wt % loaded implants is shown alone for clarity with the trend line added to illustrate the correlation coefficient of 0.998.

Figure 5

Pharmacokinetic analyses in mice show that implants release risperidone for greater than 56, but less than 83 days, while retaining coherence past the delivery period. A) Risperidone serum concentrations from mice that received implants show no detectable drug at 83 days, consistent with in vitro release patterns. B) Residual risperidone content in implants following removal from mice indicate that implants have no detectable drug at 83 days, consistent with serum concentration at the same time point shown in panel A. Additionally, these data indicate that implants can be removed at a time point beyond the period during which drug is released. However, because PLGA implants are fully biodegradable, they would not require removal as would be the case for minipumps and other non-degradable formulations. The observation that implants removed at 56 days contain 10 wt % risperidone suggests that in vivo degradation and release is faster than the rate observed in vitro. Black bars are EO sterilized implants and gray bars are surface sterilized implants.

Figure 6

Behavioral testing in mice demonstrates that bioactivity of risperidone is retained when delivered from PLGA implants. A) Risperidone implants did not alter startle amplitude. B) However, implants marginally increased PPI relative to control implant animals (p = 0.052) on 14 and 21 days post implantation. C) Risperidone implants increased the P20 amplitude in C57Bl/6J mice (p = 0.03) 28 days following implantation, indicating that chronic risperidone administration from implants achieve a comparable biological effect as previously demonstrated using another antipsychotic medication, olanzapine, in osmotic minipumps (Maxwell et al 2004).