Neuroprotective efficacy and pharmacokinetic behavior of novel anti-inflammatory para-phenyl substituted diindolylmethanes in a mouse model of Parkinson's disease

Abstract

There are currently no registered drugs that slow the progression of neurodegenerative diseases, in part because translation from animal models to the clinic has been hampered by poor distribution to the brain. The present studies examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM) compounds that display anti-inflammatory and neuroprotective efficacy in vitro. We postulated that the pharmacokinetic behavior of C-DIM compounds after oral administration would correlate with neuroprotective efficacy in vivo in a mouse model of Parkinson's disease. Pharmacokinetics and metabolism of 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5), 1,1-bis(3'-indolyl)-1-(phenyl)methane, 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1 mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and urine. C-DIM compounds given orally displayed the highest area under the curve, Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics of the compounds tested. Oral bioavailability of each compound ranged from 6% (C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for 14 days, a model of progressive neurodegeneration with a strong neuroinflammatory component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure to MPTP and probenecid prevented further loss of dopaminergic neurons in the substantia nigra pars compacta and striatal dopamine terminals, indicating that these compounds could be effective therapeutic agents to prevent neurodegeneration.

Fig. 1.

Structure and molecular weight of C-DIM compounds.

Fig. 2.

Analytical determination of C-DIM compounds in plasma by liquid chromatography–tandem mass spectrometry (LC-MS/MS). (A) Product ion spectra and proposed structures for each C-DIM compound identified by LC-MS/MS. (B) Blank mouse plasma spiked with 25 ng/ml of naringenin, the internal standard (A). C-DIM 5, 7, 8, and 12 were spiked into blank plasma at 1 ng/ml with internal standard (B). A representative mouse plasma sample spiked with 25 ng/ml naringenin internal standard (C). The solid line represents the summed m/z transitions for each C-DIM compound. The retention times for C-DIM5, C-DIM7, C-DIM8, and C-DIM12 were 3.0 minutes, 3.0 minutes, 2.8 minutes, and 3.0 minutes, respectively. The dashed line represents the internal standard for the m/z transition 271.0 → 119.0 (R_t = 1.9 minutes).

Fig. 3.

Plasma pharmacokinetic distribution of C-DIM compounds. Plasma concentrations for C-DIM compounds and relationship between the route of exposure and plasma concentration. Points represent mean plasma level for either intravenous (red) or oral gavage (black) of C-DIM5 (A), C-DIM 7 (B), C-DIM8 (C), or C-DIM12 (D) over a period of 12 hours (n = 3 animals per time point for each route of exposure) data are expressed as mean ± S.E.M.

Fig. 4.

Brain pharmacokinetic distribution of C-DIM compounds. Brain tissue concentrations for C-DIM compounds and relationship between route of exposure and concentration in brain. Points represent mean brain tissue level for either IV (blue) or oral gavage (black) of C-DIM5 (A), C-DIM7 (B), C-DIM8 (C), or C-DIM12 (D) over a period of 12 hours (n = 3 animals per time point for each route of exposure); data are expressed as mean ± S.E.M.

Fig. 5.

Tissue accumulation and metabolism of C-DIM compounds after oral administration. (A) Mice were administered a single oral dose (10 mg/kg) of C-DIM5, C-DIM7, C-DIM8, or C-DIM12 and examined for tissue distribution of each C-DIM compound 4 hours after administration, the time at which peak plasma and brain concentrations of drug were measured in pharmacokinetic studies. Colors denote different C-DIM compounds, error bars represent standard deviation, and significance is indicated by * (P < 0.05, n = 3 animals per group). Plasma (B) and urine (C) samples were also collected and treated with β-glucuronidase or saccharic acid (control), and the concentration of each C-DIM parent compound was determined by liquid chromatograpjy–tandem mass spectrometry. Mice were housed in metabolic cages for urine sample collection. Colors denote different C-DIM compounds, error bars represent standard deviation, and significance is indicated by *** (P > 0.001, n = 3 animals per group); data are expressed as mean ± S.E.M.

Fig. 6.

Metabolic scheme for C-DIM compounds. Identified metabolites of parent C-DIMs reported in Table 3 show the common oxidative metabolism observed for C-DIM5, C-DIM8, and C-DIM12.

Fig. 7.

C-DIM compounds protect against progressive loss of TH-positive neurons in the SN after subacute exposure to MPTP and probenecid. Representative montage images of TH-positive neurons in the SN indicate that, compared with saline (SAL)-treated controls (A), MPTPp treatment causes loss of dopaminergic cell bodies and processes by 7 days (B) that progresses by 14 days (C), even after cessation of MPTPp treatment on day 7. (A–C) Progressive loss of dopamine neurons expressing TH in the SN during MPTPp treatment regimen. (D–F) The SN of C-DIM–treated animals given daily oral gavage (50 mg/kg) of C-DIM5, 8, or 12 on days 7–14, after administration with MPTPp. Dopamine neuronal cell bodies and axons are preserved in these animals at levels similar to those in MPTPp 7 days treatment, indicating protection against loss of TH-positive cells when C-DIMs are administered after the onset of neuronal injury. Montages were reconstructed from each series of 20× individual images. TH immunofluorescence images were converted to inverted monochrome for presentation. Scale bar = 500 μm.

Fig. 8.

Neuroprotective effects of C-DIM compounds on TH expression in the ST. Representative montage images of TH immunostaining in the striatum of mice treated with saline (SAL) (A), MPTPp 7 days (B), MPTPp 14 days (C), MPTPp 14 days plua C-DIM5 (D), MPTPp 14 days plus C-DIM8 (E), and MPTPp 14 days plus C-DIM12 (F). Loss of TH intensity in the ST was evident 7 days after treatment with MPTPp and continued to decrease through 14 days after MPTPp treatment. All three C-DIM compounds prevented this progressive decrease in striatal TH intensity on days 7–14. Representative montages were reconstructed from each series of 10× individual images stained for TH immunofluoresence and converted to inverted monochrome for presentation. Scale bar = 500 μm.