Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Neuropharmacology. 2020 May 29;174:108152. doi: 10.1016/j.neuropharm.2020.108152

Carisoprodol Pharmacokinetics and Distribution in the Nucleus Accumbens Correlates with Behavioral Effects in Rats Independent from Its Metabolism to Meprobamate

Theresa M Carbonaro 1, Vien Nguyen 1, Michael J Forster 1, Michael B Gatch 1, Laszlo Prokai 1
PMCID: PMC7465490  NIHMSID: NIHMS1603457  PMID: 32479814

Abstract

Carisoprodol (Soma®) is a centrally-acting skeletal-muscle relaxant frequently prescribed for treatment of acute musculoskeletal conditions. Carisoprodol’s mechanism of action is unclear and is often ascribed to that of its active metabolite, meprobamate. The purpose of this study was to ascertain whether carisoprodol directly produces behavioral effects, or whether metabolism into meprobamate via cytochrome P450 (CYP450) enzymatic reaction is necessary. Rats were trained to discriminate carisoprodol (100 mg/kg) to assess time course and whether a CYP450 inhibitor (cimetidine) administered for 4 days would alter the discriminative effects of carisoprodol. Additionally, pharmacokinetics of carisoprodol and meprobamate with and without co-administration of cimetidine were assessed via in-vivo microdialysis combined with liquid-chromatography–tandem mass spectrometry from blood and nucleus accumbens (NAc). The time course of the discriminative-stimulus effects of carisoprodol closely matched the time course of the levels of carisoprodol in blood and NAc, but did not match the time course of meprobamate. Administration of cimetidine increased levels of carisoprodol and decreased levels of meprobamate consistent with its interfering with metabolism of carisoprodol into meprobamate. However, cimetidine failed to alter the discriminative-stimulus effects of carisoprodol. Carisoprodol penetrated into brain tissue and directly produced behavioral effects without being metabolized into meprobamate. These findings indicate that understanding the mechanism of action of carisoprodol independently of meprobamate will be necessary to determine the validity of its clinical uses.

Keywords: Carisoprodol, pharmacokinetics, metabolism, distribution, drug discrimination, rat

1. Introduction

Carisoprodol (N-isopropylmeprobamate, Soma®) is a frequently prescribed muscle relaxant that was first marketed in the late 1950s. It is commonly used for relief of acute musculoskeletal pain, especially for lower-back pain (Fass, 2010; Luo et al., 2004). In the last two decades, an increase in reports of abuse, dependence, and adverse effects has occurred in the US and globally (Reeves et al., 1997; Logan et al., 2000; Bailey and Briggs, 2002; Ni et al., 2007; Reeves and Burke, 2010; Nebhinani et al., 2013). In 2009, 2.9 million people reported consuming carisoprodol for non-medical purposes in the US (Substance Abuse and Mental Health Services Administration, 2011). Carisoprodol use has been associated with delusions, seizures, withdrawal symptoms, and lethality, especially in elderly patients (Bramness et al. 2007; Gonzalez et al., 2009a; Reeves and Burke, 2010). In animal models of tolerance and withdrawal, carisoprodol produced an addiction potential similar to that of other abused sedatives, producing discriminative stimulus effects similar to benzodiazepine or barbiturate compounds (Gonzalez et al., 2009b), and producing tolerance measured by loss of righting reflex and precipitated withdrawal following chronic exposure (Gatch et al., 2012). Considering the recent increase in abuse, the incidence of severe side effects, and the abuse liability led carisoprodol to be classified as a Schedule IV compound of the Controlled Substances Act (Drug Enforcement Agency, 2011).

Although carisoprodol has been widely used as a muscle relaxant, the mechanism of action is not well understood. Carisoprodol is predominantly metabolized to the active metabolite meprobamate (Miltown, Equanil; Olsen et al., 1994; Dalen et al., 1996). Previously, the effects of carisoprodol have been attributed to meprobamate, which has barbiturate-like activity at the GABAA receptor (Rho et al., 1997). Interestingly, more recent evidence suggests that carisoprodol binds to and stimulates the GABAA receptor independent from its metabolite meprobamate (Gonzalez et al., 2009b; Kumar et al., 2015).

The GABAA receptor is a ligand-gated ion channel, with several binding sites for both orthosteric and allosteric modulation, including the barbiturate, benzodiazepine, and neurosteroid sites. In a series of electrophysiological experiments, carisoprodol directly activated and allosterically modulated GABAA receptors, suggesting that carisoprodol produces a barbiturate-like positive allosteric effect not dependent on metabolism to meprobamate (Gonzalez et al., 2009b). Subsequent work indicated that carisoprodol does not act directly at the benzodiazepine, barbiturate, or neurosteroid sites on the GABAA, but may have a novel allosteric binding site (Kumar et al., 2015). In agreement with the electrophysiological data, carisoprodol produces barbiturate-like behavioral effects in rats, such that the discriminative stimulus effects of carisoprodol are similar to the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate, and were blocked by the barbiturate-site antagonist bemegride, but not by the benzodiazepine-site antagonist flumazenil (Gonzalez et al., 2009b).

The discriminative stimulus effects of ethanol are largely mediated by GABA and glutamate receptors in the nucleus accumbens (NAc) (Allen et al., 2016; Besheer et al., 2009), although pathways from other brain regions including the medial prefrontal cortex, insula, amygdala and others contribute to mediation of ethanol’s effects (Hodge and Cox, 1996; Jaramillo et al., 2016). The NAc is well-known to mediate reward and reinforcing effects via the release of dopamine and the resultant activation of mesocorticolimbic pathways (e.g., Ikemoto and Panksep, 1999). Alcohol and other GABAergic drugs of abuse indirectly cause dopamine release, thereby triggering the reward/reinforcement pathways (e.g., Koob and Volkow, 2010).

Although these electrophysiological studies confirm that carisoprodol can act directly on GABAA receptors, they do not address the extent carisoprodol enters the brain from the circulation, or whether it must be transformed into meprobamate to produce its behavioral effects. If carisoprodol is merely a prodrug for meprobamate, both compounds should have the same indications. However, meprobamate is currently prescribed for anxiety, whereas carisoprodol is prescribed as a muscle relaxant. If carisoprodol produces effects independently of its conversion to meprobamate, then there is a need to understand its mechanism of action for its clinical applications, for its abuse liability, and for treatment of overdose and dependence.

The purpose of this study was to address the following questions, 1) to what extent carisoprodol enters the brain, specifically the NAc, after systemic administration; 2) does blockade of carisoprodol metabolism to meprobamate alter its pharmacokinetics or decrease its ability to produce discriminative stimulus effects; and 3) does the time course of the discriminative stimulus effects of carisoprodol follow the time course of blood or brain levels of carisoprodol, or those of meprobamate? To address these questions, we used both in vivo microdialysis sampling at high temporal resolution from the circulation and the NAc combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Prokai et al., 2016) to assess pharmacokinetics and drug distribution, as well as drug discrimination to evaluate behavioral effects. To characterize whether carisoprodol produces effects independent from meprobamate, treatment with cimetidine, an inhibitor of the cytochrome P 450 (CYP450) enzyme, was used to impede carisoprodol’s metabolism to meprobamate as reported in previous studies (Dalen et al., 1996; Park et al., 2005). By administering carisoprodol alone and in combination with cimetidine, we measured levels of carisoprodol and meprobamate in rat blood and brain (NAc). NAc was chosen since the discriminative stimulus effects of other GABAA-ligands are mediated largely in the NAc (Allen et al., 2016; Besheer et al., 2009). Additionally, carisoprodol discriminative stimulus effects were assessed after 4-day treatment of cimetidine. If behavioral effects of carisoprodol were primarily mediated via meprobamate, blocking carisoprodol metabolism would alter carisoprodol-trained appropriate responding. Finally, a time course of the discriminative stimulus effects of carisoprodol was conducted.

2. Material and methods

2.1. Animals

Male Sprague–Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). They were housed individually and were maintained on a 12:12 light/dark cycle (lights on at 7:00AM). Body weights were maintained at 320–350 g by limiting food to 20 g/day, which included the food received during training sessions. Water was freely available in the home cages. All housing and procedures were in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2011), and were approved by the University of North Texas Health Science Center Animal Care and Use Committee.

2.2. Drugs

Carisoprodol was obtained from PCCA (Houston, TX) and was suspended in 2% methylcellulose. Cimetidine was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in 0.9% saline.

2.3. Drug discrimination procedures

Standard behavior-testing chambers (Coulbourn Instruments, Allentown, PA) were connected to IBM-PC compatible computers via LVB interfaces (Med Associates, East Fairfield, VT). The computers were programmed in MED-PC IV (Med Associates, East Fairfield, VT) for the operation of the chambers and collection of data.

Rats were trained to discriminate carisoprodol (100 mg/kg, i.p.) from 2% methylcellulose by using a two-lever choice methodology with a 20-min pretreatment time. Dose and pretreatment time were based on earlier studies (Gonzalez et a., 2009b). Food (45 mg food pellets; Bio-Serve, Frenchtown, NJ) was available under a fixed-ratio 10 schedule of reinforcement when responding occurred on the drug-appropriate lever for up to 200 lever presses or 20 pellets. There were no consequences scheduled for incorrect responses. Half of the rats were trained with drug on the right lever; half were trained with drug on the left lever. Training sessions occurred in a double alternating fashion (D-D-S-S-D, etc.), and tests were conducted between pairs of identical training sessions (i.e., between either two saline or two drug-training sessions). Animals received at least 60 training sessions in total before use in any behavioral experiment. Animals were selected for use in experiments when they had achieved 85% injection-appropriate responding for both the first reinforcer and for the total session during nine of their last ten training sessions.

The carisoprodol dose–effect relationship was tested in 10 rats 20 min following administration of vehicle, 10, 25, 50, and 100 mg/kg, i.p., respectively. A repeated-measures design was used such that each rat received all doses on separate test sessions. Doses were administered in increasing order. To assess carisoprodol-associated time course effects, a group of 8 rats received carisoprodol (100 mg/kg i.p.) and were placed in the test chambers at 10, 20, 40, 80, and 160 min after drug administration. A repeated-measures design was used, such that each rat was tested at each of the pretreatment time points. In contrast with training sessions, both levers were active, such that 10 consecutive responses on either lever led to reinforcement. Data were collected until 20 food pellets were obtained, or for a maximum of 20 min. Rats were tested only if they had achieved 85% injection-appropriate responding for both the first reinforcer and total session during the two prior training sessions. At least 3 days elapsed between test sessions.

To test the effects of cimetidine, a different cohort of rats (n=9) trained to discriminate carisoprodol received i.p. injections (2 ml/kg in 0.9% saline) of 100 mg/kg cimetidine daily for four days. On the fifth day, administration of carisoprodol occurred 20 min prior to the start of the test session. In test sessions prior to the 4-day cimetidine treatment, independent control sessions of vehicle and 100 mg/kg carisoprodol were conducted.

2.4. In vivo microdialysis

Intracranial in vivo microdialysis of rats was performed according to our earlier publication (Prokai et al., 2016) and separately from the behavioral assessment. Briefly, sampling relied on CMA/12 probes (CMA Microdialysis, Torshamnsgatan, Sweden) inserted through indwelling guide cannuale implanted 5–7 days before by stereotaxic surgery into the NAc (coordinates in mm relative to bregma: anteroposterior +1.3, mediolateral +1.6, dorsoventral −3.2; Paxinos and Watson, 1986). In addition, another microdialysis probe [CMA 20 Elite with shaft length of 20 mm and fitted with 20 kDa cut-off poly(arylether sulfone) membrane, 4 mm length x 0.5 mm o.d.; CMA Microdialysis, Torshamnsgatan, Sweden] was implanted under anesthesia into the jugular vein of the animal on the day of the experiment for a simultaneous microdialysis sampling from the blood. NAc and jugular vein microdialysates were collected using a standalone Raturn apparatus for awake animals (BASi®, West Lafayette, IN, USA) by perfusion of the probes with an artificial cerebrospinal fluid (150 mM Na+, 3.0 mM K+, 1.4 mM Ca2+, 0.8 mM Mg2+, 1.0 mM HPO42− and 155 mM Cl−) and anticoagulant solution (3.5 mM citric acid, 7.5 mM sodium citrate and 13.5 mM dextrose), respectively, at 2.0 µl/min (Prokai et al., 2004). After the animal recovered from anesthesia, both the intracranial probe (CMA/12, also from CMA Microdialysis) and the jugular probe (CMA/20) were attached to separate syringe pumps (1-ml BeeStinger; BASi, West Lafayette, IN); then, the animal was placed in a Raturn® Sampling/Caging System (MD-1404, BASi) and the probes were equilibrated by perfusion for 1 h before i.p. injection of the drug(s).

To test the effects of cimetidine, rats (n=4) previously trained to discriminate carisoprodol received i.p. injections (2 ml/kg in 0.9% saline) of 100 mg/kg cimetidine daily for four days before the start of the experiments described below.

Microdialysates were collected from both probes simultaneously every 5 min in the first hour after the final drug administration, and then every 20 min for 2 h by using a refrigerated two-channel fraction collector (HoneyComb, BASi). Placement of the cerebral probes was verified by microinjecting dyes through the guide cannula after sacrificing the animals at the conclusion of the experiments, followed by removal of the brains and sectioning them on a commercial rat brain slicer matrix for visual inspection.

2.5. Assay for carisoprodol and meprobamate

The collected microdialysates were analyzed by atmospheric-pressure chemical ionization (APCI) LC-MS/MS as described previously (Prokai et al., 2016) and using calibrations with known concentrations of carisoprodol and meprobamate dissolved in artificial cerebrospinal fluid and in the anticoagulant solution to determine their concentrations in the intracranial and jugular microdialysates, respectively, using diethyl acetamidomalonate as an added internal standard. The system consisted of a Surveyor HPLC pump equipped with a Micro AS autosampler and connected to an LTQ linear ion trap mass spectrometer (Thermo, San Jose, CA, USA). The manufacturer’s Xcalibur (Thermo, version 2.0) software was used to control acquisitions and perform data processing. After injecting 5 µL of the sample solution, the analytes were separated on a Discovery HS C18 50 mm x 2.1 mm column with 5 µm sorbent particles (Supelco, Bellefonte, PA, USA) under isocratic conditions at 0.25 mL/min with water/acetonitrile/acetic acid 68:32:0.5 (v/v/v) as a mobile phase. The APCI source of the mass spectrometer was operated in positive-ion mode with vaporizer and heated capillary temperatures set to 350 °C and 175 °C, respectively. The discharge current was maintained at 4 µA with the sheath gas and auxiliary sweep gas (both nitrogen) flow rates regulated to 25 units and 5 units, respectively. Carisoprodol was detected by MS/MS selected reaction monitoring with the protonated molecule (m/z 262.2) isolated at unit-mass width, 20% relative collision energy used for fragmenting, and m/z 176.1 chosen as product ion. Using precursor selection of m/z 218.6 ± 1.5 and 20% relative collision energy, meprobamate and diethyl acetamidomalonate were fragmented in a single MS/MS scan event with m/z 158.1 and m/z 176.1 monitored as product ions, respectively. Unbound drug concentrations were calculated from collection efficacies estimated though in vitro experiments and from subsequent measurements of carisoprodol and meprobamate concentrations in the collected samples (Prokai et al., 2016).

2.6. Data analysis

Drug discrimination data are expressed as the mean percentage of responses on the drug-appropriate lever for the first reinforcer in each test period. Response rates were expressed as a function of the number of responses made divided by the total session time. Response rate data were analyzed by one-way, repeated measures analysis of variance. Effects of individual doses were compared to the appropriate control value by using a priori contrasts. Criterion for significance was set a priori at p<0.05. Graphs for the percentage of drug-appropriate responding and response rate were plotted as a function of dose or pretreatment time. Error bars show standard error of the mean. Full substitution was defined as ≥80% drug-appropriate responding (DAR). The potency of carisoprodol that fully substituted was calculated by fitting straight line to the dose–response data by means of Origin 2019 (OriginLab Corporation, Northampton, MA). The straight line was fitted to the linear portion of the curve, defined by doses producing 20% to 80% of the maximal effect, including not more than one dose producing <20% of the maximal effect and not more than one dose producing >80% of the maximal effect. Other doses were excluded from the analyses.

From the results of the microdialysis studies, temporally-resolved drug concentrations in the neuronal membrane were estimated from the interstitial fluid (ISF) concentrations measured by LC-MS/MS: CNM(t) ~ P · CISF(t), where CNM(t) and CISF(t) are the membrane and interstitial fluid concentrations at t time after carisoprodol administration, respectively, and P is the membrane-ISF partition coefficient which was approximated with the n-octanol/water partition coefficient (Hansch and Leo, 1971). The logarithm of n-octanol/water partition coefficient (logPo/w) for carisoprodol and that of meprobamate were taken from http://www.drugbank.ca/drugs/DB00395 and http://www.drugbank.ca/drugs/DB00371, respectively. Time courses were analyzed using repeated-measures analysis of variance (ANOVA). Differences between group effects at specific time points or intervals were analyzed by t-tests with p<0.05 considered statistically significant.

3. Results

3,1. Drug discrimination

Dose–Response Study.

As shown in Figure 1, carisoprodol dose-dependently increased drug-appropriate responding from <1% to a maximum of 99.9% following 100 mg/kg (ED50=46.5 mg/kg). Response rate was not different from vehicle control [F(4,32)=2.585, p=0.056].

Figure 1. Discriminative stimulus effects of carisoprodol.

Figure 1.

Open in a new tab

Top panel shows percentage of responses (average ± standard error) made on the drug-appropriate lever as a function of dose (n=10). Bottom panel shows average (± standard error) rate of responding in responses per second (r/s). Ctrl indicates the vehicle and training drug control data.

Time Course.

The discriminative stimulus effects of carisoprodol had a rapid onset, with 75±16% drug-appropriate responding observed by 10 min following administration (Fig. 2). The peak of 100% drug-appropriate responding was observed at 40 min, with full substitution occurring between 20 and 40 min (85±12 to 100%). Drug-appropriate responding dropped to 48±17% at 80 min and to 30±13 by 160 min. Response rate was not different from vehicle control [F(5,35)=2.311, p=0.065].

Figure 2. Time course of the discriminative stimulus effects of carisoprodol.

Figure 2.

Open in a new tab

Top panel shows percentage of responses (average ± standard error) made on the drug-appropriate lever as a function of time (n=8). Bottom panel shows average (± standard error) rate of responding in responses per second (r/s). Ctrl indicates the vehicle control data.

Cimetidine.

Cimetidine failed to alter the discriminative stimulus effects of carisoprodol. As shown in Table 1, administration of vehicle produced no drug-lever responding, whereas both carisoprodol (100 mg/kg) alone and carisoprodol following four days of cimetidine treatment produced full substitution (>80% drug-appropriate responding). There was an overall effect of treatment [F(2,16)=69.36, p>0.001], with the vehicle control different from both drug groups. The carisoprodol alone and carisoprodol + cimetidine groups were not different. There was no effect of treatment on response rate [F(2,16)=1.404, p=0.274].

Table 1. Discriminative stimulus effects of carisoprodol alone and with cimetidine.

DAR = drug appropriate responding.

Condition DAR Rate
Vehicle control 0.0±0.0 0.670±0.098
Carisoprodol alone 83.7±11.2 0.765±0.128
Carisoprodol + Cimetidine 91.3±8.7 1.117±0.304
Open in a new tab

3.2. Pharmacokinetics and drug distribution

Administration of 100 mg/kg i.p. carisoprodol produced small increases in blood levels of carisoprodol [F(19,19)=3.325, p=0.006] as shown in Fig. 3. Table 2 shows peak levels of carisoprodol and meprobamate across treatments. Carisoprodol levels peaked within 30 min and remained stable for two hours. In contrast, much higher levels of meprobamate were observed [F(19,19)=18.67, p<0.001]. The levels of meprobamate peaked at 2 h after administration and were decreasing at the end of the observation period. Cimetidine co-administration increased blood levels of carisoprodol between 20 and 45 min (p<0.05). The blood level of carisoprodol was 2.74±0.35 ng/mL following cimetidine co-administration versus 1.53±0.07 ng/mL without cimetidine co-administration. In contrast, cimetidine decreased the levels of meprobamate for 45 to 60 min after administration and then decreased the peak level of meprobamate from 20.4±1.4 to 14.3±0.2 at 2 h after administration (p<0.05).

Figure 3. Unbound drug concentrations in the circulating blood after i.p. injection of carisoprodol (100 mg/kg): with and without cimetidine.

Figure 3.

Open in a new tab

Charted data are averages in ng/mL showing standard error; solid lines: carisoprodol levels, dashed line: meprobamate levels; squares: cimetidine co-administered, triangles: without cimetidine. Asterisks indicate statistically significant difference (p < 0.05) when the time courses of concentrations with and without cimetidine pretreatment were compared; arrows show direction of change. Cimetidine increased peak levels of carisoprodol and decreased peak levels of meprobamate.

Table 2.

Peak levels of carisoprodol and meprobamate

Source Treatment Carisoprodol Concentration (ng/mL) Meprobamate Concentration (ng/mL)
Blood Carisoprodol alone 1.53±0.07 20.4±1.4
C + Cimetidine 2.74±0.35 14.3±0.2
Interstitial Fluid Carisoprodol alone 3.08±0.12 18.7±1.3
C + Cimetidine 5.15±0.20 7.80±1.00
Nucleus Accumbens Carisoprodol alone 1.49±0.06 0.43±0.03
C + Cimetidine 2.49±0.10 0.18±0.02
Open in a new tab

As shown in Figure 4a, following administration of carisoprodol, the concentration of carisoprodol in interstitial fluid (CISF) peaked in NAc by 20 min and gradually tapered over time [F(19,19)=4.13, p=0.002]. Carisoprodol CISF in the NAc reached a plateau between 20 and 120 min after injections, and the peak with cimetidine co-treatment (5.15±0.20 ng/mL) was higher than that of carisoprodol alone (3.08±0.12 ng/mL), t=8.96, p<0.001. CISF of meprobamate in the NAc rose more slowly than did the levels of the corresponding carisoprodol CISF, peaking around two hours [F(19,19)=5.91, P<0.001]. Treatment with cimetidine resulted in a large decrease in meprobamate CISF in the NAc, such that administration of carisoprodol alone produced a peak meprobamate CISF of 18.7±1.3 ng/mL, whereas carisoprodol + cimetidine produced a peak meprobamate CISF of 7.8±1.0 ng/mL (t= –6.65, p=0.003).

Figure 4. (a) Unbound drug concentrations in the interstitial fluid and (b) predicted drug concentrations in the neuronal membrane phase of the rat nucleus accumbens after i.p. injection of carisoprodol (100 mg/kg): with and without cimetidine.

Figure 4.

Open in a new tab

Charted data are averages in ng/mL showing standard error; solid lines: carisoprodol, dashed line: meprobamate; squares: cimetidine co-administered, triangles: without cimetidine. Asterisks indicate statistically significant difference (p < 0.05) when the time courses of concentrations with and without cimetidine pretreatment were compared; arrows show direction of change. Cimetidine increased peak levels of carisoprodol and decreased peak levels of meprobamate.

However, neuronal cell-membrane concentrations (CNM) of carisoprodol predicted from CISF values and n-octanol/water partition coefficients (Po/w) were much higher between 10 min and 2 h than predicted CNM levels of meprobamate in this time interval (Fig. 4b). CNM of carisoprodol in NAc reached a plateau between 20 and 120 min after administration (including a spike at 60 min) [F(19,19)=4.13, p=0.002]. Again, co-administration of cimetidine caused an increase in carisoprodol’s CNM from 1.49±0.06 nM without cimetidine to 2.49±0.10 nM (t=8.96, p<0.001). There was a continuous increase in the CNM of meprobamate during the first 2 hours [F(19,19)=5.91, p<0.001]. Again, co-administration of cimetidine caused a decrease in meprobamate CNM from 0.43±0.03 nM without cimetidine to 0.18±0.02 nM (t= –6.65, p=0.003).

4. Discussion

Until recently, the behavioral effects of carisoprodol were widely believed to be produced by its active metabolite, meprobamate. Based on previous reports (Dalen et al., 1996; Park et al., 2005), the CYP450 enzyme inhibitor, cimetidine should impede the conversion of carisoprodol into meprobamate. In the present experiment, carisoprodol (100 mg/kg, i.p.) produced discriminable stimulus effects within ten minutes, peaked at 40 min and mostly dissipated by 160 min. Cimetidine (100 mg/kg, i.p.) given daily for four days failed to inhibit the discriminative stimulus effects produced by 100 mg/kg i.p. of carisoprodol. Response rate failed to show significant change from carisoprodol control. These results suggest that carisoprodol can produce discriminative stimulus effects directly without being metabolized to meprobamate. However, these behavioral data did not provide confirmation that meprobamate levels had been affected by the cimetidine treatment regimen.

Intracerebral in vivo microdialysis allows for the measurement of unbound drug concentrations in the interstitial fluid of the sampled brain region (CISF), whereas the microdialysis probe implanted into the jugular vein sampled unbound drug concentrations in the circulating blood (Tsai 2003). In the present experiment that employed high temporal resolution of sampling (Prokai et al., 2016) practically unattainable through the use of the conventional blood collection method applied to pharmacokinetics in rats (Chan, 2000), carisoprodol blood levels were significantly lower than those of meprobamate over the entire three-hour experiment. Cimetidine co-administration appeared to increase levels of carisoprodol in the first 60 min and decrease levels of meprobamate, confirming that carisoprodol to meprobamate metabolism by CYP2C19 (Dalen et al., 1996; Park et al., 2005) occurred systemically.

In the NAc, CISF of carisoprodol peaked in NAc interstitial fluid by 20 min, plateaued and then gradually tapered, whereas meprobamate levels rose to a much higher CISF (about 3-fold) about 2 h after administration. The effects of cimetidine were much more pronounced and longer lasting in the NAc than in blood, which seems to indicate that conversion of carisoprodol to meprobamate may be pharmacologically relevant. However, the large rise in the levels of meprobamate did not occur until 90 min after administration and peaked at 2 h, whereas the behavioral effects of carisoprodol peaked at 40 min after administration and was already decreasing when meprobamate levels were rising. These findings suggest that the behavioral effects of carisoprodol are produced directly by carisoprodol and not by conversion to meprobamate.

However, it is possible that the levels of carisoprodol and meprobamate in the interstitial fluid of the NAc do not accurately measure interaction of these compounds with membrane-bound receptors. Sampling by in vivo microdialysis reflects unbound drug concentrations (Tsai 2003), whereas receptors driving neuropharmacological effects such as interaction with the GABAA receptor (Gonzalez et al., 2009a) are localized in the neuronal cell membrane (Knoflach et al., 2016). On the other hand, one should consider that most drugs that enter the brain also distribute into the interstitial fluid and into membranes of cells bathed by the interstitial fluid according to their distribution coefficient influenced by ionization (electric charge on the molecule) and lipophilicity (Fridén et al., 2007). Ionization is governed by acid-base equilibria (through pKa values) (Khojasteh et al., 2011). However, both carisoprodol and meprobamate are neutral (i.e., not charged) at physiological pH; therefore, lipophilicity (usually mimicked through n-octanol/water partitioning and expressed as the partition coefficient Po/w or its logarithm) should be the principal molecular parameter (Leo et al., 1971; Khojasteh et al., 2011) correlating with the putative equilibrium established between CISF versus drug concentration at, e.g., neuronal cell membranes (CNM) for these drugs.

Carisoprodol (with logPo/w of 2.1, http://www.drugbank.ca/drugs/DB00395) is more lipophilic than meprobamate (logPo/w of 0.7, http://www.drugbank.ca/drugs/DB00371) based on literature data. Lipophilicity reflects the affinity of a drug molecule to associate with a nonpolar lipid-rich phase such as a cellular membrane versus residing in an aqueous medium such as an interstitial fluid (Leo et al., 1971; Khojasteh et al., 2011). Also, blood-brain barrier (BBB) penetration of small molecules is optimal when their logPo/w values are in the range of 1.5–2.7, with the mean value of 2.1 (Hansch and Leo, 1979; Pajouhesh and Lenz, 2005). Carisoprodol’s logPo/w of 2.1 is a practically ideal value in this regard, while meprobamate’s lipophilicity to cross the BBB is outside the optimal range with its reported logPo/w of 0.7. Therefore, and as expected, comparison of the concentration–time profiles from our studies confirmed a better access of the injected carisoprodol to the CNS compared to its systemically formed metabolite meprobamate. Consequently, while concentration of the latter was clearly higher in the circulation (Fig. 3), carisoprodol’s CISF was higher than or comparable to that of meprobamate in the NAc for 1 to 1.5 h after carisoprodol administration (Fig. 4a).

Because in vivo microdialysis allowed for the measurement of CISF, one may also approximate CNM in the sampled brain region as the product of Po/w and CISF for carisoprodol and meprobamate obtained from our experiments (CNM ~ Po/w·CISF). Accordingly, predicted CNM levels of carisoprodol were much higher between 10 min and 2 h than predicted CNM levels of meprobamate in this time interval. In fact, the CNM levels of meprobamate were low and did not appear to increase significantly until after the discriminative stimulus effects of carisoprodol were observed. It was also of interest that the time course for CNM levels of carisoprodol closely paralleled the time course for the discriminative stimulus effects of carisoprodol. CNM levels of carisoprodol peaked between 20 and 40 min and diminished steadily over the next 2 h. Similarly, the discriminative stimulus effects of carisoprodol peaked at 40 min after administration and were mostly gone by 160 min.

Taken together, the microdialysis data from blood and NAc, as well as the drug discrimination time course suggest that carisoprodol is independently capable of producing subjective effects distinct from those of meprobamate. Meprobamate does fully substitute for the discriminative stimulus effects of carisoprodol (Gonzalez et al., 2009b), so the question of why the conversion of carisprodol to meprobamate does not prolong the time course arises. One possibility is that the conversion is too slow, which is supported by the very slow rise of meprobamate in the NAc in the present study (Fig. 4b). Another contributing factor is carisoprodol’s better access to the CNS and higher affinity to the neuronal membrane where its target receptors are localized, when compared with those of meprobamate (Fig. 3 and Fig. 4).

Although the conversion to meprobamate does not appear to prolong the discriminative stimulus effects of carisoprodol, it may prolong the adverse effects, especially at high doses of carisoprodol. Although early studies suggested that carisoprodol produced little or no withdrawal (see review in Eddy, 1969), subsequent case studies of long-term use of large doses of carisoprodol have reported a wide range of severe toxicities following withdrawal (Heacock and Bauer, 2004; Morse and Chua, 1978; Reeves et al., 2004). Two studies have reported high levels of meprobamate in patients undergoing withdrawal from carisoprodol (Littrell et al., 1993; Morse and Chua, 1978). These studies support the hypothesis that conversion of carisoprodol to meprobamate can prolong withdrawal. An attempt to develop an animal model of carisoprodol withdrawal met with difficulties in administering doses of carisoprodol large enough to result in spontaneous withdrawal due to significant levels of morbidity and mortality (Gatch et al., 2012). Future studies will need to determine whether this lethality was due to accumulating levels of meprobamate.

In summary, our studies indicate that the discriminative stimulus effects of carisoprodol may be mediated primarily through its direct positive allosteric modulation of the GABAA receptor rather than through metabolism to meprobamate because, 1) The time course of the discriminative stimulus effects of carisoprodol follow the time course of blood or NAc levels of carisoprodol, and does NOT follow that of meprobamate; and 2) Blocking metabolism of carisoprodol with cimetidine does decrease levels of meprobamate in blood and NAc, but does not alter its ability to produce discriminative stimulus effects. These findings confirm that carisoprodol acts independently from meprobamate.

Highlights:

  • Discriminative stimulus effects of carisoprodol follow the time course of its brain levels

  • Inhibition of carisoprodol’s metabolism to meprobamate does not alter discriminatory stimulus effect.

  • Behavioral assessment and pharmacokinetics collectively indicate that carisoprodol acts independently from its active metabolite meprobamate.

Acknowledgments

Funding was provided by grants from the National Institute on Drug Abuse (R01 DA022370) and the National Institute on Aging (T32 AG020494), and by The Welch Foundation (endowment BK-0031).

Footnotes

Declaration of conflicting interests The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Allen DC, Ford MM, Grant KA 2018. Cross-Species Translational Findings in the Discriminative Stimulus Effects of Ethanol. Curr Top Behav Neurosci 39:95–111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey DN, Briggs JR 2002. Carisoprodol: an unrecognized drug of abuse. Am. J. Clin. Pathol 117, 396–400. [DOI] [PubMed] [Google Scholar]
  3. Besheer J,, Grondin JJ, Salling MC, Spanos M, Stevenson RA, Hodge CW 2009. Interoceptive effects of alcohol require mGlu5 receptor activity in the nucleus accumbens. J Neurosci 29:9582–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bramness JG, Skurtveit S, Morland J, Engeland A 2007. The risk of traffic accidents after prescriptions of carisoprodol. Accid. Anal. Prev 29, 1050–1055. [DOI] [PubMed] [Google Scholar]
  5. Chan PC 2000. NTP Technical Report on the Toxicity Studies of Carisoprodol (CAS No. 78–44-4) Administered by Gavage to F344/N Rats and B6C3F1 Mice U.S. Department of Health and Human Services Public Health Service, National Institutes of Health, Bethesda. [Google Scholar]
  6. Dalen P, Alvan G, Wakelkamp M Olsen H 1996. Formation of meprobamate from carisoprodol is catalysed by CYP2C19. Pharmacogenetics 6, 387–394. [DOI] [PubMed] [Google Scholar]
  7. Drug Enforcement Administration. 2011. Schedules of Controlled Substances: Placement of Carisoprodol Into Schedule IV. Fed Regist 76, 77330–77360 [PubMed] [Google Scholar]
  8. Eddy NB, 1969. Codeine and its alternates for pain and cough relief. Ann. Intern. Med 71, 1209–1212. [DOI] [PubMed] [Google Scholar]
  9. Fass JA 2010. Carisoprodol legal status and patterns of abuse. Ann. Pharmacother 44, 1962–1967. [DOI] [PubMed] [Google Scholar]
  10. Fridén M, Gupta A, Antonsson M, Bredberg U, Hammarlund-Udenaes M 2007, In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab. Dispos 35, 1711–1719. [DOI] [PubMed] [Google Scholar]
  11. Gatch MB, Nguyen JD, Carbonaro T, Forster MJ 2012. Carisoprodol tolerance and precipitated withdrawal. Drug Alcohol Depend 12, 29–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gonzalez LA, Gatch MB, Forster MJ, Dillon GH 2009a, Abuse potential of Soma: the GABA(A) receptor as a target. Mol. Cell Pharmacol 1:180–186. [PMC free article] [PubMed] [Google Scholar]
  13. Gonzalez LA, Gatch MB, Taylor CM, Bell-Horner CL, Forster MJ, Dillon GH 2009b. Carisoprodol-mediated modulation of GABAA receptors: in vitro and in vivo studies. J. Pharmacol. Exp. Ther 329, 827–837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hansch C, Leo AJ 1979. Substituent Constant for Correlation Analysis in Chemistry and Biology Wiley, New York, ISBN 10: 0471050628 / ISBN 13: 9780471050629. [Google Scholar]
  15. Heacock C, Bauer MS, 2004. Tolerance and dependence risk with the use of carisoprodol. Am. Fam. Physician 69, 1622–1623. [PubMed] [Google Scholar]
  16. Hodge CW, Cox AA 1998. The discriminative stimulus effects of ethanol are mediated by NMDA and GABAA receptors in specific limbic brain regions. Psychopharmacology 139, 95–107. [DOI] [PubMed] [Google Scholar]
  17. Ikemoto S, Panksepp J 1999. The role of nucleus accumbens dopamine in motivated behavior: a unifying interpretation with special reference to reward-seeking. Brain Res Brain Res Rev 31:6–41. [DOI] [PubMed] [Google Scholar]
  18. Jaramillo AA, Randall PA, Frisbee S, Besheer J 2016. Modulation of sensitivity to alcohol by cortical and thalamic brain regions. Eur J Neurosci 44:2569–2580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khojasteh SC, Wong H, Hop CECA 2011. Drug Metabolism and Pharmacokinetics Quick Guide Springer, New York, pp. 171–174. DOI: 10.1007/978-1-4419-5629-3 [DOI] [Google Scholar]
  20. Knoflach F, Hernandez MC, Bertrand D 2016. GABA(A) receptor-mediated neurotransmission: Not so simple after all. Biochem. Pharmacol 115, 10–17. [DOI] [PubMed] [Google Scholar]
  21. Koob GF, Volkow ND 2010. Neurocircuitry of addiction. Neuropsychopharmacology 35:217–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kumar M, González LA, Dillon GH 2015. Assessment of subunit-dependent direct gating and allosteric modulatory effects of carisoprodol at GABAA receptors. Neuropharmacology 97, 414–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leo A, Hansch C, Elkins D 1971. Partition coefficients and their uses. Chem. Rev 71, 525–616. [Google Scholar]
  24. Littrell RA, Sage T, Miller W 1993. Meprobamate dependence secondary to carisoprodol (Soma) use. Am. J. Drug Alcohol Abuse 19, 133–134. [DOI] [PubMed] [Google Scholar]
  25. Logan BK, Case GA, Gordon AM 2000. Carisoprodol, meprobamate, and driving impairment. J. Forensic Sci 45, 619–23. [PubMed] [Google Scholar]
  26. Luo X, Pietrobon R, Curtis LH, Hey LA 2004. Prescription of nonsteroidal anti-inflammatory drugs and muscle relaxants for back pain in the United States. Spine 29,E531–E537. [DOI] [PubMed] [Google Scholar]
  27. Morse RM, Chua L 1978. Carisoprodol dependence: a case report. Am. J. Drug Alcohol Abuse 5, 527–530. [DOI] [PubMed] [Google Scholar]
  28. National Research Council. 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. The National Academies Press, Washington, D.C. [Google Scholar]
  29. Nebhinani N, Aggarwal M, Mattoo SK, Basu D 2013. Carisoprodol: an underrecognized drug of abuse in north India. Gen. Hosp. Psychiatr 35, 89–92. [DOI] [PubMed] [Google Scholar]
  30. Ni K, Cary M, Zarkowski P 2007. Carisoprodol withdrawal induced delirium: A case study. Neuropsychiatr. Dis. Treat 3, 679–82. [PMC free article] [PubMed] [Google Scholar]
  31. Olsen H, Koppang E, Alvan G, Mørland J 1994. Carisoprodol elimination in humans. Ther. Drug Monit 16, 337–340. [DOI] [PubMed] [Google Scholar]
  32. Park EJ, Cho HY, Lee YB 2005. Effect of cimetidine and phenobarbital on metabolite kinetics of omeprazole in rats. Arch. Pharm. Res 28, 1196–1202. [DOI] [PubMed] [Google Scholar]
  33. Pajouhesh H, Lenz GR 2005. Medicinal chemical properties of successful central nervous system drugs. NeuroRx 2, 541–553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paxinos G, Watson C 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed, Academic Press, San Diego. [Google Scholar]
  35. Prokai L, Fryčak P, Nguyen V, Forster MJ 2016. Mass spectrometric analysis of carisoprodol and meprobamate in rat brain microdialysates. J. Mass Spectrom 51, 900–907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Prokai L, Prokai-Tatrai K, Zharikova AD, Nguyen V, Perjesi P, Stevens SM Jr. 2004. Centrally acting and metabolically stable thyrotropin-releasing hormone analogues by replacement of histidine with substituted pyridinium. J, Med, Chem 47, 6025–33. [DOI] [PubMed] [Google Scholar]
  37. Reeves RR, Beddingfield JJ, Mack JE, 2004. Carisoprodol withdrawal syndrome. Pharmacotherapy 24, 1804–1806. [DOI] [PubMed] [Google Scholar]
  38. Reeves RR, Burke RS 2010. Carisoprodol: Abuse potential and withdrawal syndrome. Curr. Drug Abuse Rev 3, 33–38. [DOI] [PubMed] [Google Scholar]
  39. Reeves RR, Pinkofsky HB, Carter OS 1997. Carisoprodol: a drug of continuing abuse. J. Am. Osteopath. Assoc 97, 723–724. [DOI] [PubMed] [Google Scholar]
  40. Rho JM, Donevan SD, Rogawski MA 1997. Barbiturate-like actions of the propanediol dicarbamates felbamate and meprobamate. J. Pharmacol. Exp. Ther 280, 1383–1391. [PubMed] [Google Scholar]
  41. Substance Abuse and Mental Health Services Administration. 2011. Drug Abuse Warning Network, 2008: National Estimates of Drug-Related Emergency Department Visits; Rockville, MD. [PubMed] [Google Scholar]
  42. Tsai TH 2003. Assaying protein unbound drugs using microdialysis techniques. J. Chromatogr. B 797, 161–173. [DOI] [PubMed] [Google Scholar]

RESOURCES