Preparation,Evaluation And Pharmacodynamics Of Dopamine Receptor Agonist Extended-release Microspheres

Parkinson’s disease（PD）,also known as paralysis agitans,is a common degenerative disease of central nervous system in middle-aged and elderly people.Clinically,there are mainly static tremor,limb and trunk stiffness,slow movement and other motor symptoms.For the treatment of PD,levodopa is the most commonly used and effective drug.However,in the long-term treatment of levodopa,PD patients will have motor complications,which becomes an urgent problem in the process of PD treatment.Therefore,some scholars have proposed continuous dopaminergic stimulation（CDS）,a new concept of PD treatment,which can provide patients with the best clinical effect and reduce the occurrence of motor complications to the greatest extent.However,the existing CDS treatment methods usually have some disadvantages such as high cost,difficult medication operations,poor patient compliance and so on.Therefore,it is necessary to develop a more effective,less invasive and better compliance CDS treatment.As a complex disease,the clinical characteristics of PD also include non-motor symptoms,among which pain is a common non-motor symptom of PD.PD patients may experience different types of pain,including akathisia,radicular/neuropathic,musculoskeletal,central pain and dystonic pain,among them the most common mainly manifested as musculoskeletal pain.This kind of pain is related to ankylosis,dyskinesia or dystonia in PD patients,which can make muscles or joints stretch continuously and produce inflammatory lesions,thus leading to inflammatory pain.PD patients are mostly elderly,often accompanied by osteoarthritis,rheumatism and other diseases,these diseases which also have inflammatory pain.Pain can significantly affect the quality of life of PD patients.Studies have found that PD can promote the release of a variety of proinflammatory cytokines such as tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）,accelerate the degeneration and apoptosis of neurons,and thus cause serious damage to the central and peripheral nervous system.Elderly PD patients themselves also have chronic low-level systemic inflammation due to‘inflammatory aging’.All in all,PD is associated with inflammatory response,and related anti-inflammatory therapy has been proved to alleviate the inflammatory state of neurons in the brain of patients with neurodegenerative diseases.Dopamine receptor agonists are drugs that are similar to dopamine in molecular structure and can directly stimulate postsynaptic dopamine receptors.Dopamine receptor agonists used in early PD therapy can delay the application of levodopa.It is the first choice for PD early treatment.As an adjunctive therapy to levodopa in patients with advanced PD,the dosage of levodopa can be reduced,thus reducing the occurrence of motor complications.Ropinirole（ROP）is a new non-ergoline dopamine receptor agonist,which can selectively stimulate dopamine D₃ and D₂ receptors in the striatum to restore the function of dopamine in PD patients.It is a first-line drug in the treatment of PD.At present,ROP is only available in common tablets and sustained-release tablets,which are usually taken multiple times a day or once a day.Since PD patients need long-term medication,it is necessary to make a more strict medication plan for the PD patients,but the delay of medication will lead to the appearance of symptoms such as inconvenient movement.At the same time,PD patients are usually accompanied by dysphagia and other characteristics,thus reducing the safety and compliance of medication.Rotigotine（RTGT）is also a non-ergoline dopamine receptor agonist,mainly through the stimulation of D₃/D₂/D₁ receptors to play an anti-PD role.Luye Pharma Co.,Ltd.prepared Rotigotine extended-release microspheres（RTGT-MS,LY03003）for injection by emulsion/solvent evaporation method using poly（lactide-co-glycolide）（PLGA）as carrier materials,which can continuously delivery drugs and stabilize the drug concentration in plasma and brain striatum.RTGT-MS can significantly increase the contralateral rotation behavior of 6-OHDA induced PD rats,and the efficacy is relatively stable.RTGT-MS combined with levodopa can effectively reduce or improve the occurrence of motor complications in PD rats.This preparation is currently in Phase III clinical trials.It is expected to become the first extended-release preparation in the world to truly achieve continuous dopaminergic stimulation in the treatment of PD.In order to research and develop extended-release microspheres of dopamine receptor agonist with clinical value,ROP and RTGT were used as representative drugs in this study.Two aspects of research work have been carried out.The first is preparation of ROP extended-release microspheres,further systematic and in-depth study of the release mechanism,pharmacodynamics,pharmacokinetics and their relationship of this extended-release preparations were researched to provide support for the preparation process research,industrial production and clinical application of this kind of drugs.Secondly,study the pharmacodynamics and mechanism of non-motor symptoms pain and inflammation of RTGT-MS which has been fnished systematic preclinical research and clinical research on the treatment of PD motor symptoms.This provides an experimental basis for the more scientific clinical application and expansion of this kind of extended-release preparations.（1）The study on preparation and characterization of dopamine receptor agonists ROP extended-release agentsIn order to improve the therapeutic effect of ROP and avoid the problems of poor safety and compliance in the application of ROP oral tablets.Based on the research and development of RTGT-MS,an extended-release microspheres of ROP（ROP-MS）were developed.The formulation composition,preparation process,in vitro drug release characteristics and in vivo pharmacokinetics of ROP-MS were studied,and the effect of ROP-MS anti-motor symptoms of PD was further investigated.In this study,ROP extended-release microspheres（ROP-MS）were prepared by O/W emulsion/solvent evaporation method with PLGA as carrier materials.The effects of the formulation and preparation process of the microspheres,such as PLGA type,PLGA concentration,drug loading,emulsifier concentration,anti-sudden release additive,the volume ratio of oil phase to water phase and emulsifying stirring speed,on drug loading,encapsulation efficiency,particle size and in vitro release of microspheres were investigated,and the optimal formulation and preparation process of ROP-MS were obtained.The preparation process is simple and repeatable.The prepared microspheres have a drug load of 21.42%,an encapsulation efficiency of 85.68%,a particle size of105.7μm,and an in vitro drug release of up to 21 days.In the study of ROP-MS accelerated release and release mechanism,the effects of ion concentration,p H value,temperature and ethanol concentration of release medium on the accelerated release of ROP-MS were investigated.The accelerated release conditions related to long-term release of ROP-MS were determined.The conditions of accelerated release associated with long-term drug release of ROP-MS were determined and the correlation between accelerated release and long-term release in vitro was establlished.The mechanism of ROP-MS release in vitro was revealed by the change of surface morphology and Ritger-Peppas model of microspheres during long-term release.The results showed that the release process of ROP-MS could be accelerated by 0.05 mo L·L^-1p H 7.4 PBS containing 5%ethanol at 37°C,which was similar to the long-term release curve with good correlation（R²=0.9980）.The in vitro release of ROP-MS was controlled by the dissolution diffusion of ROP and the gradual erosion of microspheres,and the erosion mechanism of skeleton was the main one.A HPLC-MS/MS method was established for determination of ROP in rats serum.The serum drug concentrations in rats after intramuscular injection of ROP-MS and ROP-suspensions were measured respectively to obtain drug serum concentration-time curves,and the pharmacokinetic parameters were calculated.By comparing the pharmacokinetic parameters such as T_1/2 and MRT of the two preparations,ROP-MS has obvious sustained-release effect in vivo of rats,and the release was stable within 3 weeks.The PD rat model was established by intraperitoneal injection of rotenone suspensions.The anti-PD effects of ROP-MS were evaluated by rats body weight,the scores of climbing rod test,open field test,swimming test and the number of tyrosine hydroxylase（TH）neurons in substantia nigra of rats.The results showed that the weight of rats in ROP-MS group increased gradually,and the body weight and behavioral scores of ROP-MS group were significantly higher than those of model group and ROP-suspension group on the 10th day after administration.The loss of TH neurons in substantia nigra was significantly reduced and the number of TH neurons was significantly increased.The results showed that ROP-MS can obviously improve the motor symptoms of PD rats,has a certain protective effect on dopaminergic neurons and anti-PD effect.（2）The study on analgesic and anti-inflammatory effects and mechanism of extended-release RTGT-MS of dopamine receptor agonists.In order to investigate the effect of dopamine receptor agonist extended-release preparations on non-motor symptoms of PD patients,the analgesic effect of RTGT-MS currently in clinical trials on inflammatory pain model,the influence on chronic and acute inflammation and the possible analgesic and anti-inflammatory mechanisms were studied.Intramuscular injection of RTGT-MS has obvious analgesic effect on carrageenan induced inflammatory pain in rats.RTGT-MS can reduce the levels of cyclooxygenase-2（COX-2）and Prostaglandin E₂（PGE₂）in inflammatory tissue of rats with inflammatory pain,and decrease the expression of cyclic adenosine monophosphate（c AMP）and protein kinase A（PKA）.Intraperitoneal injection of chlorpromazine hydrochloride（CPZ）can reduce the downregulation of COX-2 and c AMP induced by RTGT-MS,and partially inhibit the downregulation of PKA.It is suggested that the analgesic mechanism of RTGT-MS may be associated with peripheral D₂ receptors,as well as AC-c AMP-PKA pathways.The results of isobolographic analysis and interaction index showed that RTGT-MS combined with celecoxib has obvious synergistic analgesic effect on inflammatory pain.The combined use of the two drugs is expected to relieve the motor symptoms of PD patients,at the same time,reduce the pain of non-motor symptoms of PD patients and the pain associated with rheumatism,osteoarthritis and other diseases.The effects of RTGT-MS on inflammation were observed by cotton-pellet-induced granuloma and carrageenan induced paw edema models in rats.The expression of interferonγ（IFN-γ）,TNF-αand IL-1βin inflammatory tissues was measured by ELISA to reveal the possible anti-inflammatory mechanism of RTGT-MS.The results showed that RTGT-MS significantly inhibited the formation of cotton-pellet-induced chronic granuloma in rats,and can significantly inhibit carrageenan induced paw edema rate,significantly reduce the pathological score of rats with inflammatory plantar tissue,and inhibit pathological damage.The inhibitory effect of RTGT-MS on acute inflammation is related to reducing the expression of IFN-γ,TNF-αand IL-1βin inflammatory tissue.However,CPZ can reduce the inhibitory effect of RTGT-MS on inflammation by blockage dopamine receptor,which indicates that dopamine receptor pathway plays an important role in the anti-inflammatory of RTGT-MS.In conclusion,ROP-MS prepared in this study has high drug loading and entrapment efficiency,sustained and stable drug release in vivo and in vitro,can significantly improve the motor symptoms of PD rats,and has a certain protective effect on dopaminergic neurons.Through intramuscular injection,the drug delivery period of ROP-MS is obviously prolonged,in accord with the new concept of CDS treatment PD at present.It can solve the problems of dysphagia in PD patients,improve the safety of medication,increase patient’s compliance.RTGT-MS,a dopamine receptor agonist extended-release preparation has long-term analgesic effects on inflammatory pain and anti-inflammatory effects.It also has definite synergistic analgesic effect with non-steroidal anti-inflammatory and analgesic drug celecoxib.As an extended-release preparation for continuous dopaminergic stimulation in the treatment of PD motor symptoms,it can also reduce the pain of non-motor symptoms and the pain associated with rheumatism,osteoarthritis and other diseases of patients and reduce inflammation.This study provides a solid experimental basis for the clinical application and expansion of of this kind of extended-release preparations.