#expbio ASPET Blogging: Modified dextromethorphan displays antidepressant-like effects in the forced swim test

Figure 1: Mmmm, cough syrup. 
(Disclaimer: cough syrup is *not* 
a treatment for depression.) 

(Source: @aerial_m)

Remember that thick purple cough syrup you took as a child? Maybe you still take it when you’re so sick you feel like you’re hacking up a lung. One of the ingredients to suppress cough in some syrups is dextromethorphan (DM). It has been in use for approximately 50 yrs and has a low side effect profile. In addition to other effects, DM may act similarly to ketamine, a dissociative anesthetic that blocks NMDA receptors and was recently found to have fast-acting antidepressant properties. Because most current antidepressants take weeks to provide symptom relief, both drugs are good candidates for improved depression treatments. However, DM has fewer side effects than ketamine. Anna Scandinaro is an undergraduate student at West Virginia University in the lab of Dr. Rae Matsumoto, who is now Dean of the College of Pharmacy at Touro University in California. Anna and her co-mentor, Linda Nguyen, a MD/PhD student in Dr. Matsumoto’s lab are investigating a modified DM molecule in the treatment of depression. 

DM activates the sigma-1 receptor (σ₁R), a receptor in the brain for which the function has not yet been fully determined. However, the Matsumoto lab has shown that DM can produce anti-depressant effects through σ₁R binding (PLoS, open access). DM can also block NMDA receptors, the target of ketamine, which are excitatory and involved in learning and memory. As mentioned in another post, the two problems with current antidepressants are that they take several weeks to provide symptom relief and many people do not respond to them. DM and ketamine may address both issues because of the quick action and because they work by completely different mechanisms than current antidepressants.

However, DM has pharmacokinetic properties (how the body breaks it down) that are not sufficient for use as it is. Having read the directions on a bottle of cough medicine, you may know that it has a short half-life of only 2-3 hours (half of the time before you take another dose). This means the body metabolizes it too quickly to be of use as an antidepressant because it prevents sufficient levels from reaching the brain. Anna hypothesized that a modified version of DM, d6-D,) that is metabolized more slowly would provide antidepressant effects. She also hypothesized that the antidepressant action would be mediated by σ₁Rs, similar to unmodified DM. Her study also examined whether the modified molecule interacts with the original receptors and proteins the same way that DM does.

For the non-pharmacologists out there, you might be wondering why metabolism matters in this context. DM enters the body and passes from the stomach to the liver right away. There, it is changed (metabolized) into dextrorphan, which produces problematic side effects. Adding a less common form of hydrogen atom, deuterium, to the DM molecule may slow the breakdown of DM into dextrorphan allowing it to stay active longer and minimizing the side effects that would limit its use as a medication. Quinidine, a blocker of the P4502D6 liver enzyme that breaks down DM into dextrorphan, could also slow the metabolism.

Figure 2: Open field test.
(Source: Stockholm Brain Institute)

Anna used three doses of the modified DM molecule, d6-DM (injected into the abdomen), to see which one would provide the best antidepressant effect. She used the forced swim test to assess its antidepressant properties. In this test, mice are placed into a deep (to them) beaker of water. The animals will try to get out of the water and then give up and just float accepting that they cannot escape. Administering an antidepressant prior to the test increases the trying-to-get-out period and with d6-DM the dose at which this effect peaked was 30 mg/kg.

Mouse locomotion was also examined using the open field test, described here, to make sure the increased activity in the water was truly due to an antidepressant effect rather than just a higher level of activity in general. They did find a significant increase in activity in mice treated with d6-DM compared with saline-treated control mice. However, there was no correlation between mobility in the swim test and stimulant activity in the open field.

Next, Anna treated groups of mice with either saline, d6-DM at two doses, or d6-DM at the two doses plus quinidine, the liver enzyme inhibitor that would further slow the metabolism of d6-DM (Table 1). When d6-DM and quinidine were combined, there was a dose-dependent, but not a significant difference in immobility in the forced swim test. However, the way the experiment was designed required the statistical tests to be run on many groups of mice, which lowers the ability to detect a true statistical difference (called a Type 1 error). Kudos to Anna and Linda for reporting their stats based on experimental design! Linda did say that if they ran a separate statistical analysis on just the quinidine + d6-DM groups and compared them to the control, the quinidine + d6-DM at the 30 mg/kg dose would be significantly different. Also important, although further slowing the metabolism of d6-DM with quinidine did not affect the antidepressant-like effects of d6-DM, it significantly reduced the stimulant effects in the open field. This reinforces the idea that the antidepressant effects of d6-DM are not dependent on its stimulant effects.

Table 1: Experimental Groups

Control	d6-DM alone	d6-DM + quinidine
Saline	d6-DM, low dose	quinidine + d6-DM, low dose
Saline + quinidine	d6-DM, higher dose	quinidine + d6-DM, higher dose

In order to determine if the antidepressant effect was mediated by activation of σ₁Rs, Anna used the σ₁R antagonists BD1063 and BD1047 to block d6-DM from binding to the receptor. She performed the forced swim test again but did not find a significant difference with either drug. This means the antidepressant effect of d6-DM is not due to σ₁R binding. Linda thinks the σ₁R may still play a role, albeit a small one, as there appears to be partial reduction of the antidepressant effects of d6-DM. It is possible that d6-DM may be using other mechanisms of action (such as NMDA receptor binding), and that both these mechanisms (and others not discussed here) are contributing to the antidepressant effects of d6-DM observed in the forced swim test such that no one antagonist can significantly inhibit the effects alone.

In future studies, the Matsumoto lab will continue to study how DM could be modified to provide antidepressant effects and identify the mechanisms that convey the therapeutic effects.

*Disclaimer: research on dextromethorphan as an antidepressant is still in the early stages. Please do not try to use cough syrup as a treatment for depression. If you are depressed, please see a medical doctor for diagnosis and treatment options.