An animal study on the effect of topically administered ambroxol for dry eye.

OBJECTIVE: To evaluate the effect of 0.2% ambroxol eye drop on tear secretion and corneal healing on a rabbit dry eye model, and to delineate potential underlying mechanisms. MATERIALS AND METHOD: A mixed mechanism dry eye model was created using 12 healthy New Zealand rabbits by excision of the main lacrimal glands, Harderian gland and nictitating membrane. Establishment of the model was confirmed by the decrease of Schirmer I and increase of corneal fluorescein staining scores. Two weeks after model creation, the rabbits were randomly and evenly divided into NaCl, 0.1% sodium hyaluronate and 0.2% ambroxol groups. Each group was administered the respective eye drops 4 times a day for four weeks. The Schirmer I test and corneal fluorescein staining were performed at two and four weeks. After four weeks of treatment, the animals were sacrificed and the conjunctiva and eyelid specimens collected. Inflammatory factors IL-8, TNF-α, and goblet cell specific mucin MUC5AC were measured by ELISA while the lid meibomian gland was evaluated by oil red O staining. RESULTS: Compared with the baseline, 2 weeks after the surgery, Schirmer I test value decreased significantly (20.35 ± 5.18 mm/5 min vs 13.95 ± 4.64 mm/5 min, p < 0.01), and the fluorescein staining score increased significantly (0.5 ± 0.6 vs 5.5 ± 1.4, p < 0.01). After four weeks of treatment, compared with the NaCl and sodium hyaluronate groups, tear secretion in ambroxol group increased significantly (p < 0.01), while the corneal fluorescence staining score decreased significantly (p < 0.01). In the conjunctival tissue, significant decrease was seen in TNF-α (p < 0.01) and IL-8 [p (unilateral) < 0.05] concentrations in ambroxol group, and significant increase in MUC5AC concentration (p < 0.01) in ambroxol group as well. The lipid content in the lid meibomian glands appeared increased after the administration of ambroxol. CONCLUSION: The present rabbit dry eye model study demonstrated potentials of topically administered 0.2% ambroxol in stimulating tear and mucin secretion, inhibiting ocular surface inflammation, promoting corneal healing, and possibly augmenting meibomian gland lipid production.

Protein unbound pharmacokinetics of ambroxol in the blood and brains of rats and the interaction of ambroxol with Polygala tenuifolia by multiple microdialysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ambroxol elevates glucocerebrosidase (GCase) activity and reduces nigrostriatal alpha-synuclein burden to better ameliorate motor function in Parkinson's disease (PD). Polygala tenuifolia is a potential alternative botanical medicine for the treatment of many nonmotor symptoms of PD commonly used in Taiwanese patients. Co-administration of these two medicines pose potential herb-drug interaction. AIM OF THE STUDY: Our hypothesis is that ambroxol and P. tenuifolia may potentially possess herbal drug synergetic effects in the blood and brain. MATERIALS AND METHODS: To investigate this hypothesis, a multiple microdialysis system coupled with validated ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for rat blood and brain samples. Experimental rats were divided into three groups: low-dose and high-dose ambroxol alone (10 mg/kg, i.v. and 30 mg/kg, i.v., respectively) and ambroxol (10 mg/kg, i.v.) pretreated with P. tenuifolia extract (1 g/kg, p.o. for 5 consecutive days). RESULTS: Ambroxol easily penetrated into the brain and reached a maximum concentration in the striatum at approximately 60 min after low- and high-dose treatment. The area under the concentration curve (AUC) ratio increased proportionally at the doses of 10 and 30 mg/kg, which suggested a linear pharmacokinetic manner of ambroxol. The brain penetration of ambroxol was approximately 30-34%, which was defined as the ambroxol AUC blood-to-brain distribution ratio (AUCbrain/AUCblood). The P. tenuifolia extract did not significantly alter the pharmacokinetics of ambroxol in the blood and brain of rats. CONCLUSION: The present study suggests that it is safety without pharmacokinetic interactions for this dosing regimen to use P. tenuifolia extract and ambroxol together.

Time-shifted co-administration of sub-analgesic doses of ambroxol and pregabalin attenuates oxaliplatin-induced cold allodynia in mice.

BACKGROUND: Oxaliplatin-induced cold allodynia is a frequent complication appearing in patients treated with this anti-tumor drug. Since, there are no clear algorithms to overcome this painful condition effectively, it is important to establish novel strategies for its treatment. AIM: In this study, the ability of pregabalin and ambroxol, used as single drugs or in combinations administered in a time-shifted manner to attenuate cold allodynia was assessed in the mouse cold plate test. The hot plate test was additionally used to assess antinociceptive properties of ambroxol in the acute, thermally-induced pain model. Locomotor activity and motor coordination of mice were also evaluated. In silico studies were undertaken to predict potential binding of ambroxol to sodium channel (Nav) subtypes whose overexpression is implicated in the development of oxaliplatin-induced neuropathic pain. KEY FINDINGS: A hyperadditive antiallodynic effect of combined sub-analgesic ambroxol and pregabalin was demonstrated in oxaliplatin-treated mice. This effect was particularly strong when these drugs were given 4 h apart. Both drugs used in combination reduced animals' locomotor activity, but they did not impair motor coordination in the rotarod test. Ambroxol did not show antinociceptive properties in the hot plate test. The molecular docking studies predicted that in mice ambroxol might bind to Nav1.6 and Nav1.9 rather than Nav1.7 and Nav1.8. SIGNIFICANCE: Time-shifted co-administration of sub-analgesic doses of ambroxol and pregabalin effectively attenuates oxaliplatin-induced cold allodynia. Molecular docking model predicts preferential binding of ambroxol to mouse Nav1.6, Nav1.9 channels. This mechanism, if confirmed in vitro, might explain pharmacological activities observed in vivo.

The contribution of mutant GBA to the development of Parkinson disease in Drosophila.

Gaucher disease (GD) results from mutations in the acid ß-glucocerebrosidase (GCase) encoding gene, GBA, which leads to accumulation of glucosylceramides. GD patients and carriers of GD mutations have a significantly higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. In this study, we used the fruit fly Drosophila melanogaster to show that development of PD in carriers of GD mutations results from the presence of mutant GBA alleles. Drosophila has two GBA orthologs (CG31148 and CG31414), each of which has a minos insertion, which creates C-terminal deletion in the encoded GCase. Flies double heterozygous for the endogenous mutant GBA orthologs presented Unfolded Protein Response (UPR) and developed parkinsonian signs, manifested by death of dopaminergic cells, defective locomotion and a shorter life span. We also established transgenic flies carrying the mutant human N370S, L444P and the 84GG variants. UPR activation and development of parkinsonian signs could be recapitulated in flies expressing these three mutant variants.UPR and parkinsonian signs could be partially rescued by growing the double heterozygous flies, or flies expressing the N370S or the L444P human mutant GCase variants, in the presence of the pharmacological chaperone ambroxol, which binds and removes mutant GCase from the endoplasmic reticulum (ER). However flies expressing the 84GG mutant, that does not express mature GCase, did not exhibit rescue by ambroxol. Our results strongly suggest that the presence of a mutant GBA allele in dopaminergic cells leads to ER stress and to their death, and contributes to development of PD.

Sonolytic debromination of ambroxol process wastewater.

In this study ultrasound was examined in terms of its effectiveness in treating wastewater containing ambroxol and process water from ambroxol synthesis. The organic bromine contents of the water samples investigated were in the 20-1200 mg l(-1) range. Ultrasound is capable to debrominate the ambroxol molecule rendering it biologically degradable. The debromination rate increases with the ultrasound intensity and the 0.4 order of the organic bromine concentration. Temperature and pH have only a small influence. Bromide ions reduce the debromination efficiency of the ultrasound. No removal of the organic carbon could be observed during ultrasonic treatment. The process water from ambroxol syntheses shows higher sonolytical debromination rates than the ambroxol model water. After extracting the process water with butyl acetate, the debromination reaction of the remaining organic bromine is considerably smaller. Argon increases the debromination under certain circumstances. The specific electric energy requirements for sonolysis vary between 7 and 920 kWh g(-1) of removed organic bromine.

Identification of CYP3A4 as the predominant isoform responsible for the metabolism of ambroxol in human liver microsomes.

1. In humans, ambroxol is metabolized to dibromoanthranilic acid (DBAA) and 6,8-dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazoli ne (DHTQ). The formation of DHTQ proceeds non-enzymatically, whereas that of DBAA requires NADPH. Studies have been performed to identify the CYP isozyme(s) involved in the formation of DBAA using human liver microsomes and microsomes expressing recombinant human CYP isozymes (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 4A11). 2. The apparent Vmax and Km for the formation of DBAA were 472+/-192 pmol/ min/mg protein and 248+/-40.6 microM respectively (mean +/- S.D., n = 3). 3. Of the recombinant CYP examined, only CYP3A4 metabolized ambroxol to DBAA. The apparent Vmax and Km were 1.42 pmol/min/pmol P450 and 287 microM respectively. 4. Among the CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, diethyldithiocarbamic acid, ketoconazole), only ketoconazole inhibited the production of DBAA (> 80%) at 1 microM and anti-CYP3A antiserum almost completely inhibited the formation of DBAA. 5. These results suggest that CYP3A4 is predominantly involved in the metabolism of ambroxol to DBAA in humans.

[The pharmacokinetics and bioequivalence of various dosage forms of ambroxol]. / Untersuchungen zur Pharmakokinetik und Bioäquivalenz unterschiedlicher Darreichungsformen von Ambroxol.

An intraindividual comparative single-dose study was carried out under carefully controlled conditions on 12 healthy volunteers in order to establish the bioavailability of trans-4-(2-amino-3,5-dibromobenzyl)-amino-cyclohexanol (ambroxol) the active principle of newly developed tablets and drops, in comparison to a commercial i.v. preparation. In an additional single-dose, cross-over study on 12 healthy volunteers the bioequivalence of ambroxol was investigated after administration of a newly developed vs. a commercial dose-equivalent, sustained-release dosage form. Following off-line derivatisation using formaldehyde to the corresponding tetrahydroquinazoline compound, ambroxol was assayed from plasma by high-performance liquid chromatography. A 2-compartment model was taken as a basis for the calculation of the plasma concentration curves and the pharmacokinetic parameters following intravenous injection of the drug. After i.v. administration, the terminal elimination half-life, the apparent volume of distribution and the total plasma clearance were determined to be 3.72 h, 1.52 l/kg and 565 ml/min, respectively. From the tablet and drop formulations the systemic availabilities were calculated to 73 and 81%, respectively; the mean transit times were determined to be 6.8 and 5.4 h, respectively. Both sustained-release dosage forms investigated are bioequivalent.

[Ambroxol, comparative studies of pharmacokinetics and biotransformation in rat, rabbit, dog and man (author's transl)]. / Speziesvergleich in Pharmakokinetik und Metabolismus von NA 872 Cl Ambroxol bei Ratte, Kaninchen, Hund und Mensch.

Pharmacokinetics and biotransformation of trans-4-(2-amino-3,5-dibromo-benzylamino)cyclohexanol hydrochloride (ambroxol, NA 872 Cl) was studied using the 14C-labelled compound. Absorption after oral administration was found to be fast and complete. Elimination half-life of radioactivity in the blood was estimated as 20--25 h in rat, dog and man and as 2 h only in rabbit. This apparent elimination half-life is governed by the disposition of acidic metabolites of NA 872. In man and rabbit radioactivity is excreted almost completely into the urine, whereas in rat and dog biliary excretion is also observed. Routes of biotransformation are similar in all 4 species. NA 872 is metabolized by phase I reactions to NA 873 and finally to dibromoanthranilic acid. Phase II reactions with the parent compound and metabolites are observed mainly in man and rabbit.

[Ambroxol, studies of biotransformation in man and determination in biological samples (author's transl)]. / Ambroxol, Untersuchungen zum Stoffwechsel beim Menschen und zum quantitativen Nachweis in biologischen Proben.

15 mg trans-4-[2-amino-3,5-dibromo-benzyl)-amino]-cyclohexanol-hydrochloride (ambroxol, NA 872) was administered i.v. and orally to healthy volunteers. The metabolic pattern in urine and plasma was similar for both routes of administration. Biotransformation reactions are straightforward, yielding two major products of phage I reactions identified as 6,8-dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydro-quinazoline and 3,5-dibromo-anthranilic acid. These metabolites as well as the parent compound are also converted to conjugates, predominantly glucuronides. Quantification of unlabelled ambroxol in biological fluids is achieved by radiochemical derivatisation with 14C-labelled formaldehyde in imitation of the biotransformation.

[Prevention and therapy of respiratory distress syndrome in premature infants by means of bromhexine metabolite VIII. Animal experimental studies on the therapeutic efficacy of bromhexine metabolite VIII in premature respiratory distress syndrome]. / Prophylaxe und Therapie des Atemnot-Syndroms bei Frühgebärenden mit Bromhexine Metabolit VIII. Tierexperimentelle Untersuchungen zur therapeutischen Wirksamkeit von Bromhexine Metabolit VIII beim Atemnotsyndrom Frühgeborener.

In preparing a clinical study 14C-labelled bromhexine metabolite VIII was applied intraamnially in animal experiments. The distribution was measured in different maternal and fetal organs by thinlayer chromatography and autoradiography. A complete placental passage was found in both directions. An organspecific accumulation in the fetal lungs could not be demonstrated.