Ambroxol,the cough expectorant with neuroprotective effects

Kristin Patzwaldt,Salvador Castaneda-Vega

Αmbroxol hydrochloride (2-amino-3,5-dibromo-Nmethylbenzylamine hydrochloride) has been used as a mucolytic agent in the treatment of respiratory diseases since the late 1970s.Its effects on mucus membranes such as mucus disruption,increased mucus production,and low toxicity profile were addressed in its original German patent in 1966.These first described properties have kept Αmbroxol available worldwide and over the counter in the pharmaceutical market to this day.Since then,many mechanisms of action have been attributed to Ambroxol,including effects on autophagy,antiinflammation,and neurotrophy.This brief work dives into the latest and most compelling evidence that establishes Αmbroxol as a neuroprotective drug in neurodegenerative diseases and highlights its effects on acute neuronal injury.Ambroxol’s ability to penetrate the blood-brain barrier has provided a potential lifeline as recent studies highlight its ability to significantly improve outcomes in acute conditions such as ischemic stroke.These groundbreaking findings,which are yet to be explored in registered clinical trials,showcase Ambroxol as a potential neuroprotective agent that reduces acute injury and preserves brain function (Figure 1).

Figure 1｜Ambroxol’s neuroprotective mechanisms.

In 2020,Ambroxol was described as being able to penetrate the blood-brain barrier (BBB) even at small dosages (Yang et al.,2021) and several studies this year showed that Ambroxol can accumulate at therapeutic concentrations in the human and rat brain (Mishra and Krishnamurthy,2020;Mullin et al.,2020).This interest was fueled by the discovery that Ambroxol can increase the levels of glucocerebrosidase (GCase),an enzyme that hydrolyzes the molecule glucocerebroside into glucose and ceramide inside of lysosomes,leading to an increased cellular secretory phenotype.Ambroxol increases the expression not only of the wild-type but also of the dysfunctional mutantGBA1gene.These genes (mutant and wild-type) encode for GCase mRNΑ,as well as GCase,and increase the activity of both resulting enzyme variants (Migdalska-Richards et al.,2016;Magalhaes et al.,2018).It is not entirely understood how Ambroxol mediates these effects.Since Ambroxol acts as a chaperone molecule it has been postulated that it may stabilize GCase leading to increased lysosomal clearance.Moreover,Ambroxol can increase the levels of Cathepsin D,Lysosomal integral membrane proteins 1 and 2 (LIMP1,LIMP2)as well as the transcription factor EB,which mediate GCase transport in the endoplasmic reticulum (ER) and regulate lysosomal exocytosis.In addition,Αmbroxol has been found to accumulate in lysosomes in pneumocytes and has been associated with an overall increase in acidic vesicles and calcium release (Fois et al.,2015),which has also been observed in neurons as reduced autophagy and increased activation of the cellular secretory phenotype (Magalhaes et al.,2018).Consistent with this effect in pneumocytes,Αmbroxol treatment reduces phosphorylated α-synuclein and extracellular α-synuclein accumulations in the brain.Interestingly,α-synuclein extracellularly increases after Αmbroxol treatment even onGba1–/–cell lines,suggesting yet another independent mechanism of clearance to that of GCase activity upregulation(Magalhaes et al.,2018).Modification of GCase activity is specifically relevant to neurological diseases in which protein accumulation is a major feature,such as Gaucher’s and Parkinson’s diseases (GD and PD).In fact,currently there are 13 registered studies in www.clinicaltrials.gov involving Ambroxol and neurological diseases such as GD,PD,Lewis body dementia,amyotrophic lateral sclerosis,and diabetic neuropathy(Table 1).

Table 1｜Registration number and the title of multiple clinical trials involving Ambroxol and neurological disorders currently registered at www.clinicaltrials.com

GD is a lyposomal storage disease that occurs due to the homozygous mutation ofGBA1that leads to the production of a misfolded and dysfunctional GCase mutant enzyme in lysosomes.The overproduction of misfolded proteins produces stress on the ER,which leads to cellular dysfunction and autophagocytosis.Heterozygous or homozygous mutations ofGBA1in PD patients lead to the aggregation of α-synuclein,which results in a substantial loss of dopaminergic neurons and is the major risk factor for developing the disease in up to 15% of patients.This mutation also leads to an earlier onset of PD symptoms.Ambroxol has been shown to increase the clearance of α-synuclein and decrease autophagocytosis(Magalhaes et al.,2018),therefore leading to reduced dopaminergic cell death (Mishra and Krishnamurthy,2020).Moreover,Ambroxol can restore the activity of tyrosine hydroxylase and the activity of the dopamine transporter in rat models of PD (Mishra and Krishnamurthy,2020).Interestingly,tyrosine hydroxylase facilitates the conversion of L-tyrosine to L-DOPA,which theoretically may directly delay the onset of PD in the framework of long-term administration.

Ambroxol exhibits several beneficial effects on neuroinflammation,in the context of intracerebral hemorrhage.It reduces the number of M1-like microglia (CD16/32+cells) around the hematoma and suppresses their activation (Jiang et al.,2020).Additionally,Ambroxol diminishes the accumulation of proinflammatory cytokines,such as nitric oxide,tumor necrosis factor-α,and interleukin-1β (Jiang et al.,2020).These effects of Αmbroxol produced by the suppression of ER stress facilitate neuronal survival and reduce damage to white matter fiber bundles,thereby promoting functional recovery (Jiang et al.,2020).Ambroxol has also been shown to inhibit the production of superoxide anion,Leukotriene B4,interleukin-4,and interleukin-13 and reduces histamine release in mast cells involved in T-helper cell propagation and Th2 immune response.Αdditionally,Ambroxol has shown a reduction of Inositolrequiring transmembrane kinase endoribonuclease-1α,which is the most prominent unfolded protein response signal transducer that regulates cellular apoptotic transformation (Jiang et al.,2020).This effect is most noticeable during cellular stress,when the homeostasis of protein folding in the ER is compromised.Additionally,tumor necrosis factor receptor-associated factor 2,which mediates the transcriptional activation of nuclear factor kappalight-chain-enhancer of activated B cells and prevents apoptosis,is also underregulated following Ambroxol administration.Taken together,these specific antiinflammatory effects mean that Αmbroxol can prevent tissue damage and alter the pathophysiological cascade that leads to ischemia-induced apoptosis.

Α robust effort has been made on the applications of Αmbroxol as a neuroprotectant in neurodegenerative diseases,but few groups have explored the potential that it has for the treatment of acute neuronal injury.Currently,there are no registered studies on www.clinicaltrials.gov involving the use of Ambroxol for ischemic stroke or traumatic brain injury.We have recently shown in a 1-month longitudinal preclinical study using 53 rats that the administration of Ambroxol significantly improves ischemic stroke outcome (Patzwaldt et al.,2023).This thorough and comprehensive work by Patzwaldt et al.(2023)evaluates different and complementary stroke assessments using advancedin vivomagnetic resonance imaging (MRI),behavioral experiments,and metabolomic analysis.Using the middle cerebral artery occlusion rat model,it was demonstrated that one week of daily administration of 90 mg/kg body weight of Ambroxol significantly decreased striatal stroke volume and edema at 24,72 hours,and 1 week post-stroke.Liquefactive stroke tissue was significantly reduced in Ambroxol-treated animals in comparison to controls at 1 month poststroke onset.Moreover,behavioral outcomes were significantly improved at 24 and 72 hours post-stroke,which were consistent with maintained white matter integrity of the ipsilesional corpus callosum at 72 hours and 1 week.To further validate these results,resting state functional MRI was performed on these animals,which allowed the longitudinal evaluation of functional brain connectivity (FC) in all brain regionsin vivo.Most importantly,functional MRI analyses after stroke revealed that control animals increased the FC of the sensory-motor network while concomitantly presenting significantly worse behavioral deficits at 24 hours.On the contrary,Ambroxol-treated animals reduced the FC of the sensory-motor network while at the same time presenting significantly better performance in behavioral testing.These structural and FC findings are perfectly in line with the work of Jiang et al.(2020),who found that Ambroxol improves neuronal survival and reduces white matter damage by suppressing ER stress.The effects of Ambroxol on GBA1 expression could ameliorate the cellular stress induced by the acute insult,which results in decreased autophagy.Our work was consistent with this premise,as shown byin vivodiffusion evaluations of the stroke region and histology at 1 month.In agreement with these findings,Ambroxol has been shown to promote neuronal stem cell differentiation into neurons through the WnT/-Catenin pathway(Ge et al.,2021).Neuronal stem cells migrate and differentiate in the stroke penumbra area to restore the injured neurovascular network after injury(Hermann et al.,2014) as shown by Ge et al.(2021),who proposed yet another interesting and relevant mechanism of neuronal regeneration mediated by Ambroxol.

In addition to its anti-inflammatory effects,the effects onGBA1and cellular stress,we hypothesized that Ambroxol could act as a neuroprotectant through its effects on sodium channels,calcium homeostasis,and antioxidant properties.The functional MRI evaluations comparing baseline FC before Ambroxol and after Αmbroxol treatment demonstrated reduced connectivity of the whole brain.We interpret this effect as a general reduction of brain activity due to the inhibition of sodium channels.The reduction of cellular depolarization by blocking sodium channels could lead to a shift in energy metabolites in the brain.In fact,metabolomic analysis found increments in glucose,NΑD,ΑMP,ΑTP,3-hydroxybutarate,phosphocreatinine,ketone bodies,and several amino acids including tyrosine and valine,consistent with maintained energy reserves in the stroke region of Ambroxol animals.Interestingly,Ambroxol has been associated with an increase of mitochondrial nuclear-encoded proteins in animal models of GCase deficiency,which usually show mitochondrial dysfunction (Magalhaes et al.,2018).Therefore,a preserved mitochondrial function may also partly explain an increased energy reserve in animals treated with Ambroxol.Regarding reactive oxygen species,metabolomic analysis in the evaluation by Patzwaldt et al.(2023) also demonstrated increased reactive oxygen scavengers in the stroke area such as glutathione and pantothenate.This is also substantiated by previous studies,where Ambroxol has shown dose-dependent antioxidant activityin vitroandin vivo(Štětinová et al.,2004).Moreover,Αmbroxol has shown not only inhibition of superoxide activity extracellularly but also inhibition of their production in neutrophils and eosinophils (Duda et al.,2016).Taken altogether,Ambroxol can be used as a neuroprotective therapy also in ischemic stroke and it has the potential not only to reduce the volume of the injury but also improve functional outcomes.

Given the current strong body of evidence,therapy with Αmbroxol shows clear benefits for the treatment of neurodegenerative diseases.Interestingly,the upregulation ofGBA1results in a reduction of ER stress and as a side effect a reduction of protein aggregates.We think these multifaceted mechanisms may be beneficial not only for chronic neurodegeneration but also for acute brain injury.During acute injury the reduction of ER stress may be a defining element to avoid neuronal loss.Interestingly,currently,there are no publications or registered clinical trials (www.clinicaltrials.gov)involving Αmbroxol in stroke or traumatic brain injury.We expect however that Αmbroxol has potential in a variety of brain injury settings due to the induction of a reduced energy demand phenotype,antiinflammatory and antioxidant effects.

We encourage other groups not only to replicate the postulations in this manuscript,but also to evaluate deeper into the neuroprotective and possible prophylactic effects of Αmbroxol in acute brain insults and neurodegenerative disease.Future Ambroxol research could focus on different models of brain injury,such as traumatic brain injury,acute and chronic global hypoxia,chronic cerebral hypoperfusion,microvascular angiopathy,and epilepsy.The mechanisms described in this work could ameliorate cellular stress on the above-mentioned conditions,whether in the acute or chronic setting.For example,in epilepsy due to Αmbroxol’s effect on ion channels,it could be a relevant drug for prophylaxis of seizures and/or long-term maintenance therapy.In traumatic brain injury and microvascular angiopathy,it may act in a similar way as it does in ischemic stroke.However,some fundamental questions remain to be answered such as the mechanisms of transport of Ambroxol into the brain and the concentrations of the drug in the affected brain regions.Ambroxol concentrations in rat brains have been evaluated in naïve rats using microdialysis and ultra-high performance liquid chromatography coupled with mass spectrometry;however,these have not yet been evaluated in the disease setting.The dosages needed in different disease models may vary,especially if the BBB is interrupted.Research into Ambroxol transport mechanisms and dosis optimization in thein vivodisease setting are therefore linked and should still be carried out in order to address toxicity scenarios of high influx such as in the case of an absent BBB or,alternatively,a lack of therapeutic effect due to an intact BBB and pure reliance on possible Ambroxol transport mechanisms into the brain.Moreover,current research seems to hint that Ambroxol treatment will reduce protein aggregation in ongoing clinical trials.If these trials show improved behavioral outcomes and protein aggregate reduction in humans,then Ambroxol could be used as a tool to elucidate the relationship between protein aggregates,protein distribution patterns in the brain,time of symptom onset,and microvascular disease.

In conclusion,the current exploration of Ambroxol’s clinical impact on protein aggregates seems promising given the current state of research on its mechanisms of action.However,Ambroxol is also emerging as a promising candidate for neuroprotection in a variety of neurological disorders.

We apologize to other scientific colleagues for the possible lack of citations due to the strict reference limit.

This work was partly funded by the Clinician-Scientist grant(No.472-0-0)by the medical faculty of the University of Tübingen(to SCV).

Prior publication:We have previously published a research article titled“Repurposing the mucolytic agent ambroxol for treatment of sub-acute and chronic ischemic stroke”in the journal Brain Communications.As requested,the findings of this manuscript have been highlighted in this perspective article submission.https://doi.org/10.1093/braincomms/fcad099.

Kristin Patzwaldt,Salvador Castaneda-Vega*

Werner Siemens Imaging Center,Department of Preclinical Imaging and Radiopharmacy,Eberhard Karls University Tuebingen,Tuebingen,Germany(Patzwaldt K,Castaneda-Vega S)

Department of Nuclear Medicine and Clinical Molecular Imaging,University Clinic Tuebingen,Tuebingen,Germany (Castaneda-Vega S)

*Correspondence to:Salvador Castaneda-Vega,MD,salvador.castaneda@med.uni-tuebingen.de.

https://orcid.org/0000-0002-0484-9904(Salvador Castaneda-Vega)

Date of submission:October 6,2023

Date of decision:December 13,2023

Date of acceptance:December 23,2023

Date of web publication:January 31,2024

https://doi.org/10.4103/NRR.NRR-D-23-01664

How to cite this article:Patzwaldt K,Castaneda-Vega S(2024)Ambroxol,the cough expectorant with neuroprotective effects.Neural Regen Res 19(11):2345-2346.

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