Borneol

According to Cannapedia, haze strains like K13 are rich in borneol. Borneol has been part of Traditional Chinese Medicine for 1600 years. [1] Borneol may also be found in valerian root. The 2020 edition of Chinese Pharmacopoeia contains three commercial borneol products:

• D-borneol (Chinese name “Tianranbingpian”), extracted from fresh branches and leaves of Cinnamomum camphora, otherwise known as the camphor tree.
• L-borneol (Chinese name “Aipian”) extracted from fresh leaves of Blumea balsamifera. Wikipedia authors suggest that this camphor producing tree is more common in India and the Philippines.
• Synthetic (racemic) rborneol (Chinese name “Bingpian”).obtained via the chemical transformation of camphor and turpentine oil

Li and coauthors focused their 23 page review of the borneol literature on treatments of ischemic stroke. Authors divided the initial phase of an ischemic stroke into three components:

1. glutamate excitotoxicity
2. intracellular Ca²⁺ overload
3. oxidative stress and production of free radicals.

The authors considered inflammation/edema, necrosis/apoptosis, and blood-brain barrier damage as subacute/ secondary consequences of the eschemic stroke.[1] Reactive astrocytosis was considered as along term consequences. [1] Li and coauthors made a case for the GABAA Cl- channel as being the drug target of D-borneol mitigation of brain damage that results from an ischemic stroke.

This post will review two studies demonstrating that the GABA_A Cl^– is a target of borneol. The first study came out of Australia and was performed in Xenopus oocytes that expressed the human GABA_A channel with these sub units α1β2γ 2L. [2] Fifteen years later a Chinese group expressed different isoforms of the α subunit of GABA_A in human embryonic kidney cells (HEK). The goal was to target GABA_A Cl^– channels of inter neurons in the spinal cord that are involved in neuropathic pain, [3]. This group of the ultimate goal of a treatment for neuropathic pain that would not lead to “analgesic intolerance’ and/or decrease cognitive functions. [3] This post will ignore the D-borneol fusion molecule that has multiple targets.

A pioneering study from “down under”

This 2005 study came out of the Department of Pharmacology of the University of Sydney, Australia. Westerners will recognize the medicinal plant sources of borneol in their review of the literature: valerian (Valeriana officinalis), chamomile (Matricaria chamomilla) and lavender (Lavandula officinalis). [2] Extracts these plants are used as sedatives and to promote sleep. [2]

Explanation of an initially confusing terms

Biologists all over the world know the EC₅₀ as the effective concentration needed to achieve 50% of the maximal response. The lower the EC₅₀ the more effective the compound in eliciting the stated response. These authors used terms like EC1-5, which means the amount of GABA, γ-amino butyric acid, to deliver 1-5% of the maximal GABA_A chloride channel conductance. [3]

• 

Benzodiazepine is a positive allosteric modulator of GABA_A. A positive allosteric modulator is another term non biochemists might find confusing. A modulator is something that regulates or adjusts a response. Allo comes from the Greek work allos meaning other and stereos meaning solid or object. In biochemistry allosteric means something that changes the shape.

Borneol and γ-amino butyric opening GABA_A

How much do these two agents open GABA_A Cl^– channels by themselves? if you don’t like looking at graphs, go the the summary. Otherwise, read on.

For some reason the authors answered this question in Figures 8 and 9. Figure 8 is an excellent introduction to what it means to be a positive allosteric modulator. We need 30 μM GABA to achieve 25% of the maximum GABA_A Cl^– current. If we spike the mix with 500 μM (+)-borneol we can achieve 25% of the maximum Cl^– current with only 8 μM GABA.

Moving on to Figure 9 and our second question: over 1mM (+) borneol by itself will give us about 50% of the maximum GABA Cl^– current through the GABA_A channel. [2] If we start adding GABA to this really high concentration of borneol, we never go above 150%.

(+) and (-) borneol, when GABA is moderate to high

Figure 6 examines when GABA is sufficient to elicit 15-24% of the maximum Cl^– current. Let’s call it 1/5^th to 1/4^th of the max. If our target is 500% , we need to spike sub maximal GABA with 200 μM (-) or (+) borneol to get us to 100% of the maximum Cl- current from GABA alone. Figure 7 is easy to understand. When we have enough GABA for maximum Cl^– current. Adding a large amount of (+)borneol takes us a little bit over and (-) borneol takes us a little bit under.

(-) and (+) borneol when GABA is really low

Figure 4. With enough GABA to achieve only 1-4% of the maximum, getting to the 1000 mark on the Y-axis translates to 10-50% of the maximum Cl^– current with GABA alone. Figure 5. To make things easy, let us say we have enough GABA for 10% of the max. Our target is 1000 if we want 100% of the max. We can get there with about 700 μM borneol.

Summary and what about related compounds?

The (+) and (-) isoforms of borneol only potentiate GABA currents at really low concentrations of GABA. This is the last group of columns on the left EC1-4 GABA. The chemical structures come from PubChem. The original publication [2] did not specify the stereo isoform of iso=borneol or bornyl acetate. The authors concluded that the hydroxyl group of borneol is important for modulating GABA_A‘s response to GABA.

The savvy reader might be asking if it is possible to achieve such high concentrations in a human. According to the authors

• it is typical to use 2-3 g dried valerian root in a 200 mL water extract that contains up to 320 mM boreol, [2]
• Assuming that all of the borneol is absorbed by the GI tract, the concentrations in the human could be quite large. [2]
• The authors also discussed de-esterification of borneol acetate, the principle species in valerian root extract. [2]

According to the next paper the authors may have been using the wrong α subunit of GABA_A.

A rat pain study

This study came out of the Nanjing Medical University, China. Like many other research groups, this one was examining better means of the classical methods of treating pain: opioids and non steroidal anti-inflammatory drugs. [1] Both have side effects. This post will only discuss electrophysiology experiments conducted in cultured HEK cells and rat brains.

Cell culture system, intro

1. Human embryonic kidney (HEK) cells ere transfected with genes for α1/α2/α3/α5 , β2/β3 or γ2. The γ2 subunit contained GFP and was used as a marker of the successful transfection. The individual combinations were α1β2γ2, α2β3γ2, and α3β3γ2. [3]
2. Whole–cell patch-clamp recordings of GABA-evoked currents were made at 30 °C with the holding potential of -60 mV.
3. For GABA-evoked currents, the slides were transferred to a recording chamber, and GFP-positive cells were selected to record the currents. The interval time between each different dose of GABA was controlled.

This figure basically says that α1-3 response similarly to the agonist GABA. Panel F shows GABA and borneol in the same solution for brief periods of time indicated by the horizontal lines.

The short bars at the top of the figure tell us when the drugs were present and then washed out. The tiny blip on the far left end of the α3 row is the GABA_A receptor with an α3 sub unit responding to 1 μM GABA and 0 nM borneol. The far right tiny blip of this row corresponds to 1 μM GABA and 100 nM or 0.1 μM borneol. Small blip that it is, it is a doubling of the current with GABA. alone. An enlargement of the X-axis because the superscripts are hard to read in the original publication. We never see the maximum current from the α1 sub unit. We can only guess where the effective concentration for 50% maximum current might be. From the appearance of things, GABAA receptors with α3 or α2 sub units have 10,000x affinity for (+)-borneol than those with α1 sub units.

Tissue from rats

1. Adult male Sprague Dawley (250-300g) rats
2. (+)-Borneol was dissolved in Tween 80 (final concentration ≤ 3%) plus saline
3. After anesthetized by ethyl ether, the blood was rinsed out with a saline solution. Lumbar spinal cord (L4 and L5) was rapidly removed and 350 μm cortical slices or 300 μm transverse slices were taken for electrophysiology measurements.

In the cortex 1μM diazepam more than doubled the current of just 50 μM GABA. 1 μM borneol only increased the current 40% over 50 μM GABA. In the spinal cord, both drugs potentiate GABA currents about the same.

Borneol was concluded to not have diazepam’s cognitive inhibitory effects due to lower activity on the α1 isoform that seems to predominate the rat brain. [3] This is hard to reconcile with the mRNA expression data from ProteinAtlas.org and the historic use of borneol acetate containing valerian root extract as a sedative . [2]

Looking forward

These images came from Protein Atlas. While it it true that the α1 isoform is restricted to the human brain, the spinal cord α2 isoform that Li et al [3] were targeting for neuropathic pain, is also found in the human brain. This isoform is also found in the gastrointestinal tract. This is not to disparage a truly incredible study that demonstrated nanomolar affinity of (+)-borneol for α2β2β3γ2!

A U.S. group examined data from the Allen Brain Atlas Microarray Study that included 19 genes coding for individual subunits: α(1-6), β(1-3), γ(1-3), ρ(1-3), δ, θ, ε and π. [4 ] Expression of these sub units differs by region of the brain, the individual, and the disease state. Let’s take the hippocampus formation since it produces relatively more of the α2 sub unit of the GABA_A receptor transcripts than other regions of the brain. A 200 hypothesized that the localization of cannabinoid receptors in GABAergic interneurons in the hippocampus modulates GABAergic function and hence the disruptive effects of marijuana on spatial memory and sensory processing. [5] These authors used the synthetic CB1 agonist WIN 55,212–2 and rat brain slices. Their results were consistent with a model in whcih CB1 receptors on inhibitory basket cell terminalsdecrease GABA release and thus GABA_A-mediated synaptic input onto CA1 pyramidal neuron somata. [5]

Looking forward: Could a cannabis strain high in (+)-borneol or an edible spiked with (+)-borneol allow the decreased GABA release by basket cells on pyrimid cells of the hippocampus go a little bit further?

References

1. Li Y, Ren M, Wang J, Ma R, Chen H, Xie Q, Li H, Li J, Wang J. Progress in Borneol Intervention for Ischemic Stroke: A Systematic Review. Front Pharmacol. 2021 May 4;12:606682. free article
2. Granger RE, Campbell EL, Johnston GA. (+)- And (-)-borneol: efficacious positive modulators of GABA action at human recombinant alpha1beta2gamma2L GABA(A) receptors. Biochem Pharmacol. 2005 Apr 1;69(7):1101-11 free article
3. Li, J., Zhang, L., Xu, C., Shen, Y. Y., Lin, Y. H., Zhang, Y., Wu, H. Y., Chang, L., Zhang, Y. D., Chen, R., Zhang, Z. P., Luo, C. X., Li, F., & Zhu, D. Y. (2021). A pain killer without analgesic tolerance designed by co-targeting PSD-95-nNOS interaction and α2-containning GABA_ARs. Theranostics, 11(12), 5970–5985. cificPMC free article
4. Sequeira, A., Shen, K., Gottlieb, A., & Limon, A. (2019). Human brain transcriptome analysis finds region- and subject-specific expression signatures of GABA_AR subunits. Communications biology, 2, 153. PMC free article
5. Hoffman, A. F., & Lupica, C. R. (2000). Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience, 20(7), 2470–2479. PMC free article