| Background:Cryptococcus neoformans meningitis(CNM)is a invasive fungal infection caused by Cryptococcus neoformans.CNM is in critical condition with poor prognosis and high mortality.Amphotericin B(AMPB)is the first choice for CNM treatment recommended by most guidelines,but it has a large molecular weight(924.0 g/mol),weak lipophilicity(oil-water partition coefficient 0.95),and does not easily penetrate the blood-brain barrier.The concentration of the drug reaching the brain tissue is low.Therefore,in the guidelines formulated by the World Health Organization(WHO),the Infectious Diseases Association of the United States(IDSA)and other institutions,AMPB is recommend as the core for combination medication.An international multicenter RCT study in 2009 showed that the success rate of AMPB alone in CNM treatment during the induction phase was only 41%.The clinical retrospective analysis of CNM admitted to three large tertiary hospitals in Sichuan and Chongqing showed that the effective rate of AMPB combined with fluconazole and/or flucytosine is only 55%,which is consistent with the results reported in the literature that the effective rate of combination therapy is less than 60%.The relatively low effective rate urgently requires new treatment drugs or programs to improve existing treatments.Posaconazole(POS)is a newly marketed triazole antifungal drug with a low blood-brain barrier permeability(CSF concentration<0.01-0.56 ?g/mL),and the instructions are not indicated for the treatment of meningitis.Only one study using CNM model mice showed that POS combined with AMPB can significantly reduce brain colony counts.Since most of the existing literature reports on the pharmacodynamic interaction of POS and AMPB in vitro show "irrelevant",it is speculated that the combined use of POS and AMPB may have a pharmacokinetic interaction,leading to an increase in the brain tissue drug concentration of either or one of them.Based on this,our laboratory has conducted exploratory research.Preliminary experiments have found that POS combined with AMPB can significantly increase the concentration of AMPB in brain tissue,while POS has no significant change.The results of this study suggest that POS+AMPB is expected to become a better CNM treatment plan,and clarifying the mechanism by which POS increases the concentration of AMPB in brain tissue can provide a theoretical basis for this plan,and provide reference for future CNM treatment plan optimization and new drug development.It has strong scientific significance and research value.The way drugs pass through the blood-brain barrier is divided into active transport and passive diffusion.Studies have found that AMPB is a substrate of P-glycoprotein(P-gp)through animal experiments,and POS is a proven potent inhibitor of P-gp.It is speculated that POS can increase the concentration of AMPB in brain tissue.One is that POS inhibits P-gp to reduce the efflux of AMPB from brain tissue.Because AMPB has a large molecular weight and poor hydrophilicity and lipophilicity,the main way for it to penetrate the blood-brain barrier is simple diffusion.The amount of AMPB entering the brain tissue through this diffusion method is mainly closely related to the free blood drug concentration.Both AMPB and POS have high plasma protein binding rates,90-95%and 98%,respectively.They may compete with plasma protein binding to change the free blood drug concentration.Therefore,it is speculated that the second mechanism of POS increasing the AMPB brain tissue concentration may be POS and AMPB competes with plasma protein binding,resulting in an increase in AMPB free blood drug concentration,so that more drugs can penetrate the blood-brain barrier.Based on this,this project plans to carry out the following experiments based on the completion of CNM literature analysis and CNM treatment clinical research,combined with the preliminary results of the preliminary experiment:Construct a CNM mouse model and a HPLC and LC/MS methodology system for the determination of AMPB and POS content in biological samples,and clarify the anti-cryptococcal effects of AMPB combined with POS in vivo and in vitro,and the ratio of POS+AMPB on the increase of AMPB brain tissue concentration;Experiments and cell experiments verify whether the increase in AMPB brain tissue concentration in the process of AMPB combined with POS is passive diffusion or active transport;and then through balanced dialysis,ultrafiltration experiments,surface plasmon resonance technology(SPR)and other combined molecular docking to further clarify POS Mechanisms to increase the concentration of AMPB in brain tissue;apply transcriptome sequencing technology(RNA-seq)to comprehensively analyze other possible mechanisms,and use molecular biology methods to verify.This research consists of four parts:Chapter 1:Retrospective analysis of the clinical features of cryptococcal meningitis and the effects of different treatment optionsSearch the literature with CNM as the subject heading,and analyze the co-occurrence relationship of various antifungal drugs from the perspective of bibliometrics.The case data of CNM patients in three large tertiary hospitals in Sichuan and Chongqing in the past 5 years were screened,and patients diagnosed with CNM were included.Collect information such as gender,age,clinical symptoms and anti-fungal treatment plan,and determine the curative effect based on the results of symptoms and microbiological examinations.Use descriptive statistics to sort out and analyze the clinical characteristics,treatment plans and drug resistance data of the included patients;use chi-square test to compare the efficacy of different treatment plans.The literature analysis results show that the number of CNM-themed studies is increasing year by year."Amphotericin B" and "fluconazole","Amphotericin B" and"flucytosine" have higher Co-occurrence frequency.A total of 130 patients were included in the retrospective analysis.Common clinical symptoms include headache,fever,vomiting,cough and sputum.More patients(86.2%)received the combination regimen than the single-drug regimen during the induction period,including AMPB+FLU 62(47.7%),AMPB+FLU+FC 34(26.2%),AMPB+FC 16(12.3%),AMPB Single use 18 cases(13.8%).Most of the Cryptococcus neoformans isolated from the cerebrospinal fluid of CM patients are sensitive to itraconazole,voriconazole,FLU and AMPB,but have certain resistance to FC.The AMPB-based combination medication regimen is currently the most commonly used clinical treatment regimen,and the combined treatment effect is better than single agent(p=0.0412),but the effective rate of patients receiving combination therapy is only 55%.Chapter 2:Determination of drug concentration and pharmacodynamics of amphotericin B with posaconazole in the brain tissue of a mouse model of cryptococcal meningitisThis part of the study establishes the methodology of high performance liquid chromatography(HPLC)for the determination of AMPB content in biological samples and the methodology for the determination of POS content by liquid chromatography-mass spectrometry.The established method component specificity,resolution,detection sensitivity and the recovery rates meet the requirements of biological sample testing.The clinical isolates of Cryptococcus neoformans were selected,and the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of AMPB and POS were determined by in vitro drug susceptibility tests,and the synergistic effects were studied.The fractional inhibitory concentration(FIC)index showed that the two drugs had no synergistic antibacterial effect in vitro.30 male Kunming mice were divided into normal control group,model group,AMPB group,AMPB+POS group and POS group,with 6 mice in each group.Except for the normal control group,each group was modeled by intracerebroventricular injection of Cryptococcus neoformans suspension.After the model was established,the patients were treated for 14 consecutive days,and the brain tissues were taken for HE staining and cryptococcal colonization detection.The results show that compared with the model group,AMPB,AMPB+POS and POS treatments can significantly improve brain tissue damage in mice with meningitis,and can significantly inhibit the proliferation of Cryptococcus neoformans in the brain of mice.Among the AMPB group,AMPB+POS and POS groups,the pathological damage of the AMPB+POS group was improved most obviously,and the number of cryptococcal colonization was the lowest.18 male Kunming mice were injected with Cryptococcus neoformans suspension into the lateral ventricle to create meningitis models.After modeling,they were divided into AMPB group,AMPB+POS group and POS group,with 6 animals in each group.Plasma and brain tissue were collected after 4 days of treatment,and the content of AMPB was measured by HPLC,and the content of POS was measured by liquid chromatography-mass spectrometry.The results showed that the brain tissue concentration of AMPB in the AMPB+POS group was significantly higher than that in the AMPB group alone,and there was no significant difference in the brain tissue concentration of POS compared with the POS alone group.Chapter 3:Study on the mechanism of posaconazole on the active transport of amphotericin B across the blood-brain barrierIn view of the fact that POS combined with AMPB can significantly increase the brain tissue concentration of AMPB and improve the efficacy,this part of the study is based on clarifying the effects of AMPB and POS on the expression of P-gp and Breast cancer resistance protein(BCRP),respectively At the animal and cellular levels,specific transporter inhibitors Zosuquidar(P-gp inhibitor),Ko143(BCRP inhibitor)and Elacridar(P-gp and BCRP inhibitor)are added to clarify the role of transmembrane transporters(P-gp and BCRP,etc.)in the passage of AMPB through the blood-brain barrier.The mice were divided into blank control group,AMPB group and AMPB+POS group,each with 6 mice.After 4 days of administration,qRT-PCR and immunofluorescence techniques were used to detect the expression level of the BCRP and P-gp protein and genes in the brain tissues of mice in each group.The BCECs were divided into blank control group,AMPB group and AMPB+POS(4.0 ?g/mL)group.The expression levels of BCRP and P-gp genes and proteins in cells of each group were detected by qRT-PCR and immunofluorescence technology.The results of qRT-PCR and immunofluorescence showed that AMPB or/and POS had no significant effect on the expression of P-gp and BCRP genes and protein in mouse brain tissues and BCECs cells.The mice were randomly divided into 5 groups,namely AMPB group,AMPB+POS group,AMPB+Zosuquidar group,AMPB+Ko143 group and AMPB+Elacridar group,with 6 mice in each group.After 4 days of continuous administration,the mouse plasma and brain tissue were collected,and the content of AMPB in the plasma and brain tissue was detected by HPLC.The results showed that compared with the AMPB group,POS combined with AMPB significantly reduced the concentration of AMPB in plasma while increasing the concentration of brain tissue,and also significantly increased the ratio of the concentration of AMPB in brain tissue to plasma.However,the P-gp inhibitor Zosuquidar,the BCRP inhibitor Ko143,and the dual inhibitor Elacridar of P-gp and BCRP had no significant effect on the concentration of AMPB in plasma and brain tissue.The BCECs were divided into 8 groups,AMPB,AMPB+POS(2.0?g/mL,4.0 ?g/mL and 8.0 ?g/mL),AMPB+Zosuquidar;AMPB+Ko143,AMPB+Elacridar and AMPB+serum albumin(Albumin),a total of 8 groups,37 Incubate at ?,collect cell samples at 30 min,60 min,120 min,and 180 min.HPLC method is used to detect the content of AMPB taken into the cells by BCECs.The results showed that compared with the AMPB group,different concentrations of POS,Zosuquidar,Ko143 and Elacridar had no significant effect on the uptake of AMPB by BCECs,while Albumin could significantly inhibit the uptake of AMPB by BCECs.Mice were randomly divided into three groups,namely blank Control group,amphotericin B group(AMPB),amphotericin B+posaconazole group(AMPB+POS).After continuous administration for 4 days,the heart,liver,spleen,lung,kidney,muscle and plasma were collected 4h after the last administration,and the content of AMPB in each tissue was detected by HPLC.Meanwhile,ALT,AST,TBIL,DBIL,CREAin plasma were detected.The results showed that POS combined with AMPB significantly increased the concentration of AMPB in liver,kidney and lung compared with AMPB alone.Compared with the blank control group,AMPB alone and POS+AMPB increased the damage of liver cells and the deterioration of renal function,which was manifested by the significant increase of plasma ALT,AST and CREA levels,but there was no significant difference in bilirubin levels among all groups.Compared with AMPB group alone,liver and renal function of POS+AMPB group showed no statistically significant difference.Chapter 4:Study on the passive transport mechanism of posaconazole affecting the amphotericin B across the blood-brain barrierAfter excluding the role of transmembrane transporters such as P-gp and BCRP,in the cell uptake experiment,Albumin's restriction on the uptake of AMPB by BCECs suggests that there may be a "distribution phase" interaction between the two,that is,through competition for plasma protein binding.The free drug concentration changes to increase the brain tissue concentration.Five solutions of AMPB,AMPB+2.0 ?g/mL POS,AMPB+4.0 ?g/mL POS,AMPB+8.0?g/mL POS and AMPB+16.0 ?g/mL POS were prepared with normal mouse plasma,and POS was detected by the equilibrium dialysis method The effect on the combination of AMPB and Albumin.At the same time,five groups of solutions of AMPB,AMPB+2.0 ?g/mL POS,AMPB+4.0 ?g/mL POS,AMPB+8.0 ?g/mL POS and AMPB+16.0 ?g/mL POS were prepared with normal mouse plasma and tested by ultrafiltration method.The influence of POS on the combination of AMPB and Albumin.The results of balanced dialysis and ultrafiltration experiments showed that compared with the AMPB group,POS of 4.0 ?g/mL,8.0 ?g/mL,and 16.0 ?g/mL could significantly increase the amount of free AMPB in plasma.Further use molecular docking and molecular dynamics to evaluate the binding potential and stability of POS,AMPB and Albumin,analyze their potential binding sites and amino acid residues,and simulate the binding mode between the two.The results of molecular docking and molecular dynamics showed that compared with AMPB,POS has a stronger binding potential with Albumin.The binding sites of POS,AMPB and Albumin partially overlap,and both drugs can bind to ARG218 and ASP451.SPR was used to calculate the binding rate constants and dissociation rate constants of AMPB and POS and Albumin respectively,and the POS interference experiment of AMPB and Albumin binding was also applied.The results show that the affinity of POS and Albumin is stronger than that of AMPB.The presence of POS can affect the binding of AMPB and Albumin in a concentration-dependent manner.POS can increase the amount of AMPB free drug.But POS does not affect the binding of AMPB to ?-acidic glycoprotein(AAG).Analyze other possible mechanisms by RNA-seq;divide BCECs into blank control group,AMPB group and AMPB+POS(4.0 ?g/mL)group,use RNA-seq to find the differentially expressed genes between the three groups of cells,and use Western Blot detect the protein expression levels of differential genes.RNA-seq results showed that there were 14 differentially expressed genes in the blank group,AMPB group and AMPB+POS group,and 2 genes related to tight junction proteins of the blood-brain barrier,namely EGR-1 and PDGF-B.Western Blot verification results showed that there was no significant difference in the protein expression of EGR-1 and PDGF-B in each group.Conclusion:1.Among the CNM patients in the three hospitals in Sichuan-Chongqing area,86.2%of the patients used AMPB as the core combination regimen during the induction period.Compared with the AMPB regimen alone,the combination regimen has better curative effect,but its effective rate is still less than 60%;2.AMPB and POS have no synergistic antibacterial effect in vitro.The pathological and colony count results show that compared with AMPB or POS alone,the combination of POS+AMPB in vivo has the best effect on CNM.This study proved for the first time that this increase in efficacy was achieved by the combined use of POS and AMPB,which significantly increased the concentration of AMPB in brain tissue;3.Compared with AMPB alone,POS+AMPB can significantly increase the concentration of AMPB in liver,kidney,lung,and brain tissues.The combined use of AMPB and POS to increase the concentration of AMPB in brain tissue has nothing to do with active transporters such as P-gp and BCRP existing on the cell membrane of BCECs.At the same time,no active transporters that can increase the concentration of AMPB in brain tissue were found at the level of BCECs;4.Both AMPB and POS can bind to the ARG218 and ASP451 amino acids of Albumin,and the binding capacity of POS and Albumin is stronger than AMPB.When AMPB and POS are used in combination,it can cause the dissociation of AMPB and Albumin,which increases the amount of free drug in plasma.The diffusion method increases the concentration of brain tissue through the blood-brain barrier,which is the main mechanism by which POS increases the concentration of AMPB in the brain tissue;5.POS has an effect on the transcription of blood-brain barrier-related proteins EGR-1 and PDGF-B,but the protein expression is not significant.Does POS change the permeability and transport of the blood-brain barrier by affecting protein expression and protein function in the process?The function of the protein promotes the increase of the concentration of AMPB in brain tissue.In this study,no obvious evidence has been found,but further research is needed to rule out changes in the permeability of the blood-brain barrier and the active transport mechanism.6.This study confirms for the first time that the combined use of POS and AMPB can achieve a synergistic effect in the treatment of CNM mouse models.The main mechanism is achieved by competing for plasma protein binding in the body to increase free drugs to enter the brain tissue.This study provides a theoretical basis for the increase in clinical treatment indications of POS,and provides a new target and idea for AMPB to increase its effectiveness. |
PDF Full Text Request