Synthesis And Biological Evaluation Of μ-opiate Receptors PET Radioligand ¹¹C-CFN Study On Mechanism Underlying Acupuncture Analgesia Based On Acupuncture Anaesthesia In Craniocerebral Operations

Background:Acupuncture anaesthesia is a substantial development and breakthrough of Chinese Acupuncture based on the modern scientific technology. As the capital ingredient of acupuncture anaesthesia, acupuncture analgesia (AA) has gained increased popular research at home and abroad for many years. Many studies, including neurobiochemistry, histopathology and radioligand binding assay of receptor showed that, AA was a complex physiological process mediated by various neurotransmitters, neuropeptides, modulators and receptors, which take effect by activating the pain-related structures in brain. Of which, endogenous opioid peptides and opiate receptors (ORs) are the major neurochemical substances engaged in AA. However, the direct clinical evidence of these central mechanisms is limited as they were based on animal experiments mostly. Interest in investigating acupuncture with various imaging techniques have been growing since the mid 1990s. The development of image techniques, such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET), have opened a "window" into the brain that allow us to gain an appreciation of the anatomy and physiological function involved during acupuncture in humans non-invasively, and provided the more objective and direct approach to study the point specificity and central mechanism of AA.According to characteristics of receptor-ligand binding domains, positron emission tomography (PET) receptor imaging had its superiority in real-timely investigating the distribution (localization), quantity (density) and function (affinity) of the trace receptors in vivo with non-invasive approach. It would interpret the central action mechanism of AA and promote reasonable application of acupuncture anaesthesia if the PET receptor imaging should be used in AA research. As well-known, it is necessary for receptor imaging to select the suitable radioligand. This study was a subproject of the National Program on Key Basic Research Project (973 Program), "study on mechanism underlying acupuncture analgesia based on acupuncture anaesthesia in craniocerebral operations". Its aim was to set up an automatic method for online synthesis ofμ-ORs PET radioligand 11C-carfentanil (CFN) using domestic 11C module, and to evaluate its physical, chemical and biological characteristics, and all of which were in order to conduct the clinical trial ofμ-ORs PET imaging for AA subsequently.Contents:Objective:To set up a fast and effective method for online automatic synthesis of 11C-CFN using domestic 11C module.Methods:11C-CH3I was converted to 11C-Triflate-CH3, then the latter was bubbled into a V-tube containing 0.5 mg precursor desmethyl-CFN in 0.15 ml dimethyl sulfoxide, and which was placed at room temperature (n=12), iced bath (n=2), and heating bath (550C,2 min, n=3), respectively, to obtain 11C-CFN. Sep-Pak C2 column was used for purification of the product.3 ml binary system aqueous solution (isopropanol/aqueous ammonia) was used to displace the crude product into C2 column, then the C2 column was eluted by 10 ml water thrice, dried by nitrogen gas next (10ml/min×3 min), and washed out by 0.5 ml ethanol at last.Results:The overall synthesis of 11C-CFN required approximately 20 min from end of bombardment (EOB). The synthesis yield of 0.5 mg precursor at room temperature was on average (35.5±2.2)%(n=12, uncorrected); the yield of iced bath was decreased to (5.7±2.3)%(n=2); heating bath was (34.9±3.7)%(n=3). The synthesis yield of 1.0 mg precursor at room temperature was on average (36.8±3.9)% (n=3). HPLC purification of radiolabeled compound was not required.Objective:To identify 11C-CFN by analytical HPLC, and to establish the quality control standard of 11C-CFN including its physical, chemical and biological characteristics. Safety of the product was evaluated by toxicity test on mice. Methods:The product was assayed by an analytical HPLC system (stationary phase:Xterra RP 18 column (5μm,3.9×150 mm, Waters); mobile phase:V (acetonitrile):V(H2O):V(0.02 mol/L of ammonium acetate)=55:45:0.1, pH 6.5; flow rate:0.5 ml/min; detection:u.v. absorbance at 224 nm; radioactivity by Nal crystal flow-cell). Quality control was carried out referring to United States Pharmacopoeia and "quality control guidelines of positron drugs" published by State Food and Drugs Administration. Toxicity test for 11C-CFN was performed in twelve mice that were divided into experimental and control groups randomly. Loading dose of 11C-CFN and equal volume of 10% ethanol was injected via caudal vein respectively. Reaction after injection of each mouse was observed. Then, one week of conventional breeding later, the main issue and organs of all mice were removed for pathological examination.Results:The retention time for 11C-CFN was 5.1 min, which was consistent with standard CFN. The radioactive contaminants and residual precursor were not detectable in final product by reversed phase HPLC. 11C-CFN for injection was clear and transparent, pH 6.5-7.0, radiochemical purity by HPLC was>98%, radioactive half-life was T1/2=20±2 min, radioactive concentration was 9.2 mCi/ml, specific activity was 0.75 Ci/umol, ethanol content was< 10%, permeation pressure of filter membrane was≥0.4Mpa, bacteriology and endotoxin tests were negative. The potent biological effect of CFN was validated in toxicity test on mice.Objective:To investigate the regional distribution of 11C-CFN in rats, and to evaluate the selectivity and saturability of 11C-CFN bound toμ-ORs.Methods:Twelve rats were divided into 4 groups of 3 rats per group randomly. 11C-CFN was administered intravenously in each rat via caudal vein, then the rats were killed at 5,15,30,45 min after injection respectively. A blood sample was obtained, then the brain, heart, lung, liver, spleen, kidney, and small intestine were rapidly removed. Tissue samples were rapidly weighted and counted for carbon-11 using an automatic gamma counter. Data was calculated as percent injected dose per gram tissue (%ID/g) for each tissue, and as %ID/g for each encephalic region. Furthermore, the change of radioactivity in brain with naloxone treatment was observed.Results:At 5 min after administration of 11C-CFN, radioactivity of the most tissue was up to peak value. Liver ((6.19±3.11)%ID/g) and kidney ((9.03±2.36)%ID/g) showed the highest uptake, and within 3 session afterwards, the uptake in liver was further increased but kidney. A rapid blood clearance and a high brain uptake were discovered, and brain/blood ratio of 1.82-1.39 was found from 5 to 45 min. Predominant uptake in thalamus ((4.26±0.89)%ID/g) and striatum ((4.05±1.08)%ID/g) was observed in rats brain at 5 min after injection, and the following encephalic region was cerebral cortex ((2.63±0.89)%ID/g), pons ((2.26±0.57)%ID/g), hippocampus ((2.17±0.55)%ID/g), and cerebellum ((2.15±0.39)%ID/g), respectively. Meanwhile, thalamus showed a slow clearance but cerebellum. Treatment with naloxone significantly reduced the uptake of 11C-CFN in each encephalic region, and the radioactivity was decreased to 74.3% and 78.9% of controls in thalamus and striatum respectively.Conclusions:1. This study firstly explained the radiolabeling of ORs PET radioligand 11C-CFN at home. Experimental results indicated that, automatic online synthesis of 11C-CFN using domestic 11C module has a short time of synthesis process, stable yield, and a high radiochemical purity. The major quality control outcomes met the main related standard.2. Characteristics of radiolabeled synthesis and purification for 11C-CFN in the present study compared with previous studies included:1) based on the stable synthesis yield, precursor dosage was decreased 50%(1.0 mg to 0.5 mg), as a result, economic cost was saved; 2) strong base for reaction medium was not required; 3) content of aqueous ammonia (1%,3 ml) for eluting was reduced, pH of the product was close to neutrality; 4) the use of fibrous anion exchanger for final purification was cancelled, the synthesis process was further simplified.3. Biodistribution studies performed in rats of 11C-CFN showed a rapid absorption, extensive distribution, rapid blood clearance, and a high brain uptake. A metabolic pathway via liver and kidney was found. Radioactivity in thalamus and striatum, the regions with known predominance ofμ-ORs, was accumulated markedly and cleared slowly, consistent with binding toμ-ORs selectively by CFN. Treatment with naloxone significantly blocked the 11C-CFN binding in each encephalic region, validating the saturability of 11C-CFN binding withμ-ORs. 4. In the future, contrast analysis between traditional biodistribution experiments in-vitro and animal PET imaging in-vivo was expected to carry out. Furthermore, more feasible analytical methods for receptor binding could also be studied with the help of animal PET imaging.5. CFN, aμ-ORs agonist, was reported to have a potency 7000 times that of morphine. It could induce coma and respiratory depression if the usage was inappropriate. In order to avoid pharmacological effects from 11C-CFN in human subjects, it is necessary to further improve the specific activity of final product.