Sustained Intrathecal Delivery Of Amphotericin B Using An Injectable And Biodegradable Thermogel

ObjectiveCryptococcal meningitis,caused by Cryptococcus neoformans or Cryptococcus gattii,is a fatal central nervous system(CNS)infectious disease with a worldwide distribution.It's an opportunistic fungal infection.Amphotericin B(AMB)is the first-recommended drug in guideline.However,it has many side effects that most patients can't bear and only2%-3%of AMB can cross the blood-brain barrier(BBB)by intravenous injection.Intrathecal administration makes contribution to the concentration of AMB in cerebrospinal fluid(CSF)but the neurological toxicity seems inevitable.Lumber puncture should be performed 2 to 3 times a week to supply AMB clinically which is difficult to reach the ideal pharmacokinetic level.All the characters limit AMB's effect against cryptococcal meningitis.The PLGA–PEG–PLGA triblock copolymers are a kind of mature complete thermogel with good biodegradation and biocompatibility.When the temperature is lower than the body temperature,the copolymer is liquid,which can carry the drug.When the temperature reaches the body temperature,the copolymers will become a gel state which can release drug slowly.In this paper,we proposed the PLGA–PEG–PLGA triblock copolymers to load AMB(AMB in gel)and injected into subarachnoid space by lumbar puncture.In this way,PLGA–PEG–PLGA triblock copolymers served as a temporary storage in CSF to release AMB slowly.In this way,AMB in gel can reduce the number of times of administration,decrease the neurotoxicity and side effects of AMB and solve the current treatment problems of cryptococcal meningitis.MethodsThe first part was the acquisition and the correlation property of AMB in gel.PLGA–PEG–PLGA triblock copolymers were first synthesized.The chemical structure,composition of the copolymers and MWs and MW distributions was determined by~1H-NMR and gel permeation chromatography system(GPC).Dissolve the copolymers into a different concentration of the aqueous polymer solutions with normal saline(NS).The sol–gel transition temperature was determined and select the appropriate concentration.The physical method was used to load AMB in the given concentration of polymer solution(AMB in gel).The rheological analysis of the aqueous polymer solutions and AMB in gel was performed to estimate whether AMB have effect on the aqueous polymer solutions.The second part was the in vitro experiment of AMB in gel.The cumulative release profiles of the polymer aqueous solutions which were containing 2 mg/mL AMB,4 mg/mL AMB and 8 mg/mL AMB was determined.The release data were assessed via first-order release equation.The third part was the in vivo experiment of AMB in gel.The toxicology and biocompatibility of AMB in gel were evaluated on healthy rats.AMB in gel,AMB solution,blank thermogel and NS were injected into the subarachnoid space of rats respectively.Then the survival and activity was observed every day after administration.The activity of limbs was evaluated by the modified Tarlov's scores.On the 7th,14th and21st day after administration,rats were sacrificed and the spinal cord which was near the injection site was cut.Hematoxy-Eosin(HE)staining was used to observe the morphology of spinal cord neurons.In pharmacodynamics,the rat model of cryptococcal meningitis was established.On the 7th day after infection,AMB in gel,AMB solution and blank thermogel were injected into the subarachnoid space of rats respectively.On day of 14th and 21st,CSF was taken for CSF culture and latex agglutination test(LAT)to compare treatment effects.ResultsIn the first part of experiment,the result of ~1H-NMR and GPC showed that the molecular weight(MW)was 1790-1500-1790 and the polymerization was under control.The ideal polymers were obtained.The sol–gel transition temperature was 34°C and the appropriate concentration of polymer aqueous solution was 25 wt%.There was no change in dynamic rheological analysis of polymer aqueous solution brfore and after laoding AMB which means that AMB had no effect on polymer aqueous solutions.In the second part of experiment,the cumulative release profiles suggested that AMB in gel could release AMB for 36 days.The cumulative release rate of 2 mg/mL,4 mg/mL and 8 mg/mL AMB was 89.0%?77.1%?65.6%respectively.All the release datas were in accord with the first-order equation of drug release(R~2>0.95).In the third part of experiment,the Tavlor's scores of AMB in gel group were higher than those of AMB solution group(P<0.05).The survival rate showed that AMB solution was more likely to cause death than AMB in gel(P<0.05).The HE staining of spinal cord indicated that AMB in gel group was less damage than AMB solution.The result above showed the neurotoxicity of soinal cord in AMB in gel was weaker than that of AMB solution.In the pharmacodynamic experiment,the result of CSF culture showed that the number of Cryptococcus in AMB in gel group was less than AMB solution group.LAT showed that the titer of AMB in gel group was lower than AMB solution group.These two results all indicated that the therapeutic effect of AMB in gel was better than AMB solution.ConclusionThe AMB in gel had more advantages than AMB solution.It could release AMB for more than one month.The antibacterial effect in vivo was better than AMB solution.In addition,it could decrease the neurotoxicity of AMB and reduce the number of times of administration.Thus,it was an ideal drug for treatment of Cn meningitis and also a good way of continuous intrathecal delivery.As a model disease,Cn meningitis provided reference for continuous intrathecal delivery of other disease.