Preliminary Pharmaceutical Evaluation Of The Compound Inhalation Ingredients Diallyl Disulfide And L-menthol For The Treatment Of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease(COPD),as a major worldwide health problem,is characterized by persistent airflow limitation which is usually progressive and associated with an enhanced chronic inflammatory response in the airway and the lung to noxious particles or gases.The World Health Organization(WHO)has estimated that it will be increased to being the third leading cause of death,making this a global epidemic by 2020.The pathogenesis of COPD is very complicated including chronic inflammation,an imbalance of protease/anti-protease activities,oxidative stress,apoptosis,and autoimmune mechanisms.Cigarette smoking is considered as the major cause of COPD which can induce oxidative stress,trigger pulmonary inflammation and immune dysregulation,followed by activating epithelial cells and macrophages,releasing inflammatory mediators such as tumor necrosis factor alpha(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6)and matrix metallo peptidases(MMPs),and finally leading to inflammatory process in airway,progressive airflow limitation and cell injury and apoptosis.Thus,researchers have been focusing on intervening with the inflammatory processes of COPD by inhibiting different steps of molecular and cellular pathways involved.It is plausible that agents with anti-oxidative,anti-inflammatory and immune regulation activities might offer promise for the prevention and treatment of COPD.To date,glucocorticoid has been the first-selected medicine for the treatment of COPD by systemic or inhaled medication.The newest inhalation therapies such as inhaled corticosteroids and ?2 agonists provide patients with different choices.However,inhalation of corticosteroids can increase the risk of pulmonary infection,and oropharynx fungal infection while systemic application of corticosteroids can induce systemic immunity impairment,and increase the risk of more adverse events such as infection,steroid diabetes,and osteoporosis.Therefore,researchers are committed to finding new treatments for COPD with better curative effect and lower side effects.Ingredients derived from medicinal plants and natural herbal products are often regarded as promising agents due to their anti-inflammation,antioxidant and immune system function enhancing activities.Diallyl disulfide(DADS)is the main organosulfur ingredient in garlic which has shown diverse pharmacological properties such as anti-inflammation,anti-oxidation and immune regulation activities.The anti-inflammatory effects of DADS observed in previous studies can be attributed mainly to its ability to decrease inflammatory mediators by inhibiting NF-?B translocation and i-KB phosphorylation as well as suppress the expression of mRNAs for IL-1? and TNF-?.Based on the reported studies,the mechanism by which DADS protected against oxidative stress could be concluded as inducing the activation of HO-1/Nrf2 pathway,increasing Nrf2 nuclear translocation and by reducing ROS,lipid peroxidation,Bax/Bcl-2 ratio,caspase-3 activation,and phosphorylation of JNK and p38.Since ancient times,Mentha species have been used in traditional medicines due to their antispasmodic and carminative properties.Recently,more attention has been paid on the pharmacological effects of their active constituents,such as L-menthol,which is the main component of essential oil of from Mentha species.Several studies demonstrated that L-menthol has diverted various pharmacological properties including analgesic,antibacteria,antitussive,immune modulator,anti-apoptotic,antimicrobial and antifungal activities,especially its significant anti-inflammatory and antioxidative effects.Moreover,L-menthol was identified as the main ingredient in many compound preparations which are clinically effective in several respiratory diseases such as bronchitis,allergic rhinitis,laryngopharyngitis and sinusitis in China.Moreover,diallyl disulfide(DADS)and L-menthol were also identified as the main highly volatile ingredients in a fork garlic respiratory therapy discovered in China which has shown encouraging clinical efficacy in treatment and prevention of COPD and lung cancer.But the mechanism by which this therapy could alleviate COPD is not clear.In the present work,we evaluated the effects of DADS and L-menthol on COPD from four parts:(1)the pharmacological activities of DADS and L-menthol in vitro;(2),the pharmacological activities and molecular mechanism of DADS and L-menthol in COPD rats;(3),the pharmacokinetics of DADS and L-menthol;(4),the drug-drug interactions of DADS and L-menthol.(1)The pharmacological activities of DADS and L-menthol in vitro.For the anti-inflammation studies in vitro,a lipopolysaccharide(LPS)-induced mouse macrophages Raw264.7 and CSE induced HFL-1 cells models were established.Cell survival was evaluated using sulforhodamine B(SRB)assay.Then,the effect of DADS and L-menthol on the phagocytosis of Raw264.7 cells was evaluated using neutral red assay.The levels of NO in LPS-stimulated Raw264.7 cells were determined for screening the LPS incubation time.Next,the effects of DADS and L-menthol on the levels of TNF-?,IL-1? and IL-6 induced by LPS were measured using commercial ELISA kits while the mechanism was determined using western blot assay.Briefly,we found that DADS and L-menthol inhibited LPS-induced rising NO,cell cytokines production of TNF-?,IL-1? and IL-6 in a dose-dependent manner in the non-cytotoxic concentration range of 5 to 200 ?M.Besides,western blot assay revealed that DADS and L-menthol suppressed the transcriptional activity of NF-?B p 65 and the degradation of its inhibitor i-KB while protein expression Nrf2 and NQO1 were effectually enhanced.Moreover,DADS and L-menthol could downregulate the expression of matrix metallo peptidases and tissue inhibitors of MMPs,indicating DADS and L-menthol played a role in the regulation of the imbalance between MMPs and TIMPs.(2)The pharmacological activities and molecular mechanism of DADS and L-menthol in COPD rats.For the study of the activities of DADS and L-menthol in rats,a rat COPD model was established by intraperitoneal injection of CSE.The effects of DADS and L-menthol on COPD rats were studied through intraperitoneal injection and inhalation,respectively.Briefly,the results showed a role for DADS as an anti-inflammation agent through decreasing cell influx and suppressing pro-inflammation cytokine production via inhibiting NF-?B pathway.In addition,the mechanism by which DADS exerted anti-oxidation activities can be summarized as reducing in the levels of oxidative stress markers whereas inducing in the activities of antioxidant through activating Nrf2 pathway.Moreover,MMP-9 and TIMP-1 expression were down-regulated by DADS.Furthermore,an immune regulation activity was also found in DADS by the regulation of the influx of CD4+and CD8+T cells.(3)The pharmacokinetics of DADS and L-menthol.The pharmacokinetics and bioavailability studies of DADS and L-menthol after inhalation and intravenous injection were studied.The oral bioavailability of AMSO and AMSO2 were 29.32%and 46.14%while that the inhalation bioavailability of AMSO,AMSO2 and L-menthol were 15.31%,14.21%and 50.24%,respectively.the metabolism and the enzymic kinetic study of DADS in rat erythrocytes were studied.Briefly,DADS was metabolized to AM and the Km of the apparent enzyme kinetics of rat erythrocytes was 73.88 ±9.65?M while the Vmax of the apparent enzyme kinetics of rat erythrocytes was 16.38 ± 1.00?M/min/5×108 rat erythrocytes.(4)The drug-drug interactions of DADS and L-menthol.The thorough investigation of the inhibition effect of L-menthol,DADS and its metaboites on cytochrome(CYP)P450 enzymes is essential to predict drug-drug interactions.Briefly,a human liver microsomal model was adopted to elucidate the inhibitory effect of L-menthol on CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1,and CYP3A4 using phenacetin,tolbutamide,omeprazole,dextromethorphan,chlorzoxazone and testosterone as probe drugs,respectively.The results illustrated DADS and its metabolites had a moderate inhibition on CYP2E1 while L-menthol showed a moderate inhibition on CYP2D6 and CYP1A2.This means that DADS and L-menthol may affect the disposition of medicines primarily dependent on these pathways.In a word,the pharmacological activities and molecular mechanism of DADS and L-menthol in vitro and in COPD rats as well as the biotransformation and drug-drug interactions were evaluated in the present work.Together with all the results and the reported data,DADS and L-menthol can be considered as promising low toxicity and multi-target drugs for the treatment of COPD.DADS and L-menthol have shown significant regulation function on inflammation,oxidative stress,elastic protease imbalances and immune imbalance,which are the major pathogenesis of COPD.Briefly,the compound ingredient significantly reduced the total number of white blood cells especially the number of macrophages in rats while obvoiusly suppress the release of inflammatory cytokines in mice via the mechanism of inhibiting NF-kappa B p65 signal pathway.Besides,the compound ingredient exerted anti-oxidation activities by reducing the levels of oxidative stress markers whereas inducing in the activities of antioxidant through activating Nrf2 pathway.Moreover,the morphology assessment revealed that compound ingredient was more effective than budesonide in the improvement of emphysema via the regulating of the imbalance between proteins and antiproteins.Whatsmore,DADS and L-menthol have shown a significant reduction in CD4+and CD8+T cells in the lung indicating their roles in the regulations of immune imbalance.The biological transformation and pharmacokinetic study showed that the main metabolites of DADS were AMSO and AMSO2.The Tmax of AMSO is shortened from 6 h to 2 h after inhalation compared with oral administration.The oral bioavailability of AMSO and AMSO2were 29.32%and 46.14%while they were 15.31%and 14.21%after inhlation,respectively.Drug-drug interaction results revealed that DADS and its metabolites,AMS,AMSO,AMSO2 are all moderate inhibitors of CYP2E1 while the inhibition of the metabolites were stronger than that of DADS.L-menthol was moderate inhibitor of CYP2D6 and CYP1A2.Fortunately,the compound ingredients have not shown obvious inhibitory effect.on CYP3A4 assays.