| Cryptococcal meningitis (Cryptococcal meningitis, CM) is a kind of intracranial infection caused by Cryptococcus neoformans or Cryptococcus gattii varieties. It is one of the most common opportunistic central fungal infections mainly found in human immunodeficiency virus (HIV)-positive patients, susceptible populations such as leukemia and lymphoma patients, patients with long-term use of corticosteroids and immunosuppressants, and with immune deficiency due to various reasons, however, there are still about half of the patients who had no evidence of underlying disease or immunodeficiency suffered CM. Since the1980s, there have been totally about one million HIV-associated cryptococcal meningitis patients in the world with the increasing incidence of AIDS each year. The number of the CM patients is also growing due to the increase of the application of broad-spectrum antibiotics, hormones and immunosuppressive agents, cancer chemotherapy and organ transplantation.The challenges in the treatment of CM correlate with its long course of disease, high relapse rate and unsatisfactory therapeutic efficacy. The main principles of the clinical treatment of cryptococcal meningitis is the treatment of anti-fungal treatment and the underlying disease. Emphasis was placed on combination use of antifungal agents such as polyenes, flucytosine and fluconazole. Although treatment of cryptococcal meningitis has improved, the mortality and morbidity of CM is still high. The3-month mortality rate during management of acute cryptococcal meningoencephalitis is, however, approximately20%even if with the use of anti-retroviral therapy.Amphotericin B, an important polyene antifungal, is still the first-line option in treatment of cryptococcal meningitis. It is a concentration-dependent drug, so the concentration of AMB in CSF has to be high enough to reach its minimal inhibitory concentration (MIC). However, its poor penetration into CSF, ranging from undetectable to2%-3%, even if the blood-brain barrier is damaged in intracranial infection, its penetration into CSF is no more than4%of serum concentrations, limits its effect against CM. Many clinicians choose intrathecal AMB in order to increase the drug concentration in the cerebrospinal fluid. Conventional intrathecal AMB might increase the drug concentration in CSF and improve therapeutic efficacy, however, the toxicities associated with AMB given intrathecally have limited its clinical application.We have reported a new therapy, continuous intrathecal AMB in treatment of CM. The results revealed that the AMB concentrations with continuous intrathecal therapy were significantly enhanced and maintained a stable and effective level (T>MIC). The adverse reactions and toxicity of AMB were significantly reduced, while it is common in conventional intrathecal and intravenous administration.However, there are lots of things unknown about the pharmacokinetics of AMB in cerebral spinal fluid (CSF) during continuous intrathecal administration. Further study of the pharmacokinetics of AMB in continuous intrathecal administration to guide the clinical use of this new therapy may increase the cure rate, reduce the mortality, and lighten the burden on the families and society.Part One Cisterna Magna and Intrathecal Catheterization in RabbitsObjectTo introduce a rabbit model of combining cisterna magna and intrathecal catheterization to reduce the injury of the animals, increase the success rate of catheterization, and create a proper model for the study of the pharmacokinetics of AMB in continuous intrathecal administration.Method1. Ten New Zealand white rabbits were randomly divided into two groups (n=5each), one group were used for methylene blue intrathecal injection, another were used for scanning electron microscope.2. All animals were anesthetized and cisterna magna and intrathecal catheterization were performed sequentially. Then the rabbits were checked if there were any complications during the following72h after operation.3. Methylene blue injection:Five rabbits underwent the continuous infusion of1%methylene blue solution (100ul/kg) for6h with an infuse pump via the inserted catheter. Then the animals were subsequently with10%paraformaldehyde in PBS. The position of the catheter was checked and distribution of the dye in the spinal cord and brain was assessed by autopsy.4. Scanning Electron Microscopy:morphological observations were performed with scanning electron microscopy (SEM) in five rabbits to confirm if the catheter was properly inserted into the spinal subarachnoid space or any injury of the spinal and never.Result1. Distribution of Methylene Blue:All rabbits underwent catheterization survived with no paralysis, cerebrospinal fluid leakage, bladder dysfunction (incontinence or urinary retention) and other complications. CSF were taken without blood or brain tissue except one were found with little blood in the first6h but returned normal afterwards.2. Distribution of Methylene Blue:All of the catheters were placed into the spinal subarachnoid space. The surface of the spinal cord and the brain was dyed blue, the darkest parts were located at the spinal cord near the tip of the catheter, and the farther apart from the tip of the catheter the paler the spinal cord stained. The base of the brain was also dyed, but the surface of the cerebellum and hemispheres looked pale.3. SEM Findings:The tips of all catheters were located in the subarachnoid space. SEM found that the catheters were located in the subarachnoid space without any damage in the spinal cord. There was not any clot or other exudates around the catheter.ConclusionThis method we created has a good stability, repeatability and high success probability. Moreover, no signs of spinal cord and spinal nerve root injury were found by neurological observation. The rabbit model we created in this study permits better investigation of the pharmacokinetics of continuous intrathecal AMB.Part Two Pharmacokinetics of AMB in Continuous Intrathecal AdministrationObject To provide better experimental basis for the clinical application of continuous intrathecal administration of AMB by comparing the pharmacokinetics of continuous and conventional intrathecal AMB.Method1. The ten New Zealand white rabbits were randomly divided into two groups, in which five rabbits were used for the continuous intrathecal AMB and five rabbits were used for the conventional intrathecal AMB.2. Rabbits in continuous intrathecal administration group received continuous intrathecal AMB (0.15mg/kg/24h) for72h, and CSF was taken at1,2.3,4,5,6,8,12,24,28,32,40,48,56,64and72h after the start of drug administration. Animals were checked if there were any of the complications such as fever, convulsion and paralysis during drug administration.3. Rabbits in conventional intrathecal administration group received conventional intrathecal AMB (0.015mg/kg) for30min, and CSF was taken at the same time points as continuous intrathecal administration group. Complications were also checked in this group.4. Concentrations of AMB in CSF samples were determined by the liquid chromatographic-tandem mass spectrometric assay (LC-MS/MS).Result1. Two rabbits in the conventional intrathecal injection group suffered fever and convulsion, and two revealed paralysis in the right rear limb. However, there were no adverse drug reactions, including neurological impairments in the continuous intrathecal administration group.2. There were significant differences in the pharmacokinetics of AMB between the two groups. In continuous intrathecal administration group, the AMB concentration reached the peak of3.41at1h and then decreased progressively to a concentration of0.31ug/ml at72h, while in conventional intrathecal administration group, the concentration of AMB peaked6.96ug/ml at4h and then decreased to a stable level of1.06-1.39ug/ml after24h.Conclusion1. There were no significant complications or nervous toxicity in continuous intrathecal administration while complications like fever, tremorsa and paralysis were common in conventional intrathecal injection.2. Continuous intrathecal infusion can provide a rational pharmacokinetics with stable and effective drug concentrations, contribute to a better clinical efficacy and less neurological impairments. Continuous intrathecal AMB may be a better way in treatment of cryptococcal meningitis. |
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