Real-Time Impedance Detection of Intra-Articular Space in a Porcine Model Using a Monopolar Injection Needle.

Rheumatoid arthritis and osteoarthritis can be treated through specific drug injection into the intra-articular space. Several failures during drug injection attempts with conventional fluoroscopy and ultrasonography in a small area of the intra-articular space have been reported. In this work we present an innovative impedance measurement-based method/algorithm for needle tip positioning to enhance image-guided intra-articular vaccination treatment. A novel algorithm for detecting the intra-articular space in the elbow and knee joints of a live porcine model is reported. An impedance measurement system was developed for biological tissue measurement. The electrical impedance in the intra-articular space was monitored and the needle tip was examined by ultrasonography. The contrast dye was vaccinated and checked using fluoroscopy to confirm that the dye was properly inoculated in the cavity. The electrical impedance was estimated for various needle inclusion profundity levels in saline solution, which were broadly used to evaluate the proposed device for in vivo examinations. Good efficiency was observed in the impedance-based measurements using a monopolar injection needle for intra-articular therapy. To enhance the needle tip positioning for intra-articular therapy, the intended impedance measurement device with a monopolar injection needle can be used as a complement to existing modalities.

Three new decenynol glucosides from Artemisia scoparia (Asteraceae).

Three new decenynol glucosides were isolated from the aerial parts of Artemisia scoparia. Their structures were determined to be 6E,8Z-decadien-4-yn-ol 1-O-ß-d-glucopyranoside, 6E,8E-decadien-4-yn-ol 1-O-ß-d-glucopyranoside, and 6E-decen-4-yn-ol 1-O-ß-d-glucopyranoside based on extensive spectroscopic (NMR and MS) analysis. [Formula: see text].

Lock-in Amplifier-Based Impedance Detection of Tissue Type Using a Monopolar Injection Needle.

For successful intra-articular injection therapy, it is essential to accurately position the tip of the injection needle into the target joint area while administering the drug into the affected tissue. In this study, we investigated the feasibility of a monopolar injection needle and lock-in amplifier (LIA)-based impedance measurement system for detecting the tissue type where the needle tip is located. After positioning the monopolar injection needle tip into the dermis, hypodermis, or muscle layer of pork tissue, the electrical impedance was measured in the frequency range of 10 Hz to 10 kHz. We observed a difference in the results based on the tissue type where the needle was positioned (p-value < 0.01). Therefore, the monopolar injection needle with electrical impedance measurement can be used to improve intra-articular injection therapy through non-destructive and real-time monitoring of the needle position in the tissues.

Endoscopic botulinum toxin injection for treatment of pharyngeal dysphagia in patients with cricopharyngeal dysfunction.

BACKGROUND: Dysphagia associated with neurologic disorders is a problem worldwide. However, few studies have assessed the efficacy of endoscopic botulinum toxin injections into the cricopharyngeal muscle using a pharyngoscope. METHODS: Fourteen patients who received an endoscopic botulinum toxin injection due to cricopharyngeal muscle dysfunction and a neurological disorder from 2006 to 2017 were retrospectively reviewed at a single tertiary medical center. The toxin was injected at a 100 international unit (IU) total dose under direct endoscopic vision. The success of the procedure was evaluated comprehensively using the following: clinical resolution of the previous symptoms, functional oral intake scale (FOIS) for the clinical evaluation of swallowing, and videofluoroscopic swallowing study (VFSS) to check decreased retention of barium at the pyriform sinus and the epiglottic vallecula. RESULTS: The study included 12 males and 2 females (mean age, 58 years old; range, 37-82 years). Three patients (21.4%) were confirmed clinically and eleven were confirmed by VFSS. Eleven patients (78.6%) were managed successfully with the injection. Three patients (21.4%) did not improve despite the injection. CONCLUSION: An endoscopic botulinum toxin injection may be a good treatment option for patients with dysphagia and cricopharyngeal dysfunction due to a neurological disorder. Large, prospective, long-term follow-up studies are needed in the future.

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Ultrasound versus fluoroscopy-guided cervical medial branch block for the treatment of chronic cervical facet joint pain: a retrospective comparative study.

OBJECTIVE: To compare the mid-term effects and advantages of the ultrasound (US)-guided with fluoroscopy(FL)-guided cervical medial branch blocks (CMBBs) for chronic cervical facet joint pain through assessment of pain relief, functional improvement, and injection efficiency. METHODS: Patients with chronic cervical facet joint pain who received US- (n = 68) or FL-guided CMBBs (n = 58) were included in this retrospective study. All procedures were performed using a FL or US. The complication frequencies, treatment effects, functional improvement, and injection efficiency of CMBBs were compared at 1, 3, and 6 months after the last injection. RESULTS: Both the NDI and VNS scores showed improvements at 1, 3, and 6 months after the last injection in both groups, with no significant differences between groups (p < 0.05). Furthermore, the treatment success rate at all time points was not significantly different between groups. Logistic regression analysis revealed that the injection method (US- or FL-guided), the number of injections, sex, analgesic use, and age were not independent predictors of treatment success. Compared with FL-guided CMBB, US-guided CMBB was associated with a shorter administration duration and fewer needle passes. CONCLUSIONS: Our results suggest that, compared with FL-guided CMBBs, US-guided CMBBs require a shorter administration duration and fewer needle passes, while providing similar pain relief and functional improvements. Therefore, US-guided CMBBs can be considered as an effective alternative for the conservative management of chronic cervical facet joint pain.

Voxel-based statistical analysis of brain metabolism in patients with growth hormone deficiency after traumatic brain injury.

OBJECTIVE: The aim of this study was to investigate the regional cerebral metabolism related to growth hormone deficiency (GHD) after traumatic brain injury (TBI) using F-18 fluorodeoxyglucose positron emission tomography (F-18 FDG PET) images. METHODS: Twenty-three patients with diffuse axonal injury following TBI were enrolled. They underwent brain F-18 FDG PET study and an insulin tolerance test (ITT). According to the results of ITT, they were divided into two groups: patients with GHD and subjects with TBI but normal Growth Hormone (GH). Voxel-based statistical analysis was performed and the regional cerebral glucose metabolism shown on F-18 FDG PET from 10 patients with GHD was compared with those from 13 patients without GHD. Analysis was performed using SPM2 to identify regions where decreased changes in regional cerebral glucose metabolism were significantly related to GHD. RESULTS: Compared with subjects with TBI but normal GH, patients with GHD after TBI showed decreased cerebral glucose metabolism in the Left superior frontal gyrus, Right angular gyrus, Right superior temporal gyrus, Left inferior temporal gyrus, Left anterior and middle cingulate gyrus and Right anterior and middle cingulate gyrus. (puncorrected < 0.005). CONCLUSIONS: The findings are suggestive of the brain region influenced by GHD. These cortical areas are involved in regulation of intellectual function, executive function and working memory.

Ultrasound guided intra-articular ketorolac versus corticosteroid injection in osteoarthritis of the hip: a retrospective comparative study.

OBJECTIVE: Intra-articular steroid injection has been widely used in the management of symptomatic osteoarthritis; however, its frequent use is avoided since there is an increase in the incidence of articular infection and several mechanical side effects such as cartilage breakdown and loss of elasticity of the articular cartilage. For these reasons, nonsteroidal anti-inflammatory drugs instead of corticosteroids can be considered for intra-articular injection. On this basis, we investigated the effects and safety of ultrasound-guided intra-articular ketorolac versus corticosteroid injection for patients with osteoarthritis of the hip. MATERIALS AND METHODS: This retrospective study included 98 patients with diagnoses of hip osteoarthritis who underwent ultrasound-guided intra-articular ketorolac or corticosteroid injection. Fifty patients who received ultrasound-guided intra-articular corticosteroid injection were administered a mixture of 0.5% lidocaine and triamcinolone. Forty-eight patients who received ultrasound-guided intra-articular ketorolac injection were administered 0.5% lidocaine and ketorolac. Outcome measurement was assessed using the Harris hip score and verbal numeric pain scale, which were evaluated before the injections and at 1, 3 and 6 months following the injection. Univariate analysis (using the x (2) test) and multiple logistic regression analysis were performed to evaluate the relationship between the possible outcome predictors (injected medications, patients' age, gender, pain duration and Kellgren-Lawrence classification) and the therapeutic effects. RESULTS: The Harris hip score and verbal numeric pain scale were improved at 1, 3 and 6 months after the injection in both groups. No statistical differences in the Harris hip score and verbal numeric pain scale were observed between the groups. The success rate was also not significantly different among the time periods of 1, 3 and 6 months. Multiple logistic regression and univariate analysis showed that injected medications patients' age, gender, pain duration and Kellgren-Lawrence classification were not independent predictors of successful outcome at midterm follow-up. CONCLUSION: The treatment of osteoarthritis of the hip with intra-articular ketorolac injection is as effective as that with intra-articular corticosteroid injection. Intra-articular ketorolac injection can be considered useful for patients with contraindications to using corticosteroids.

Regioselective hydroxylation of omeprazole enantiomers by bacterial CYP102A1 mutants.

A large set of Bacillus megaterium CYP102A1 mutants are known to metabolize various drugs to form human metabolites. Omeprazole (OMP), a proton pump inhibitor, has been widely used as an acid inhibitory agent for the treatment of gastric acid hypersecretion disorders. It is primarily metabolized by human CYP2C19 and CYP3A4 to 5'-OH OMP and a sulfone product, respectively. It was recently reported that several CYP102A1 mutants can oxidize racemic and S-OMP to 5'-OH OMP and that these mutants can further oxidize 5'-OH racemic OMP to 5'-COOH OMP. Here, we report that the S- and R-enantiomers of OMP are hydroxylated by 26 mutants of CYP102A1 to produce 1 major metabolite (5'-OH OMP) regardless of the chirality of the parent substrates. Although the binding of R-OMP to the CYP102A1 active site caused a more apparent change of heme environment compared with binding of S-OMP, there was no correlation between the spectral change upon substrate binding and catalytic activity of either enantiomer. The 5'-OH OMP produced from racemic, S-, and R-OMP could be obtained with a high conversion rate and high selectivity when the triple R47L/F87V/L188Q mutant was used. These results suggest that bacterial CYP102A1 mutants can be used to produce the human metabolite 5'-OH OMP from both the S- and R-enantiomers of OMP.

Ultrasound-guided versus fluoroscopy-guided sacroiliac joint intra-articular injections in the noninflammatory sacroiliac joint dysfunction: a prospective, randomized, single-blinded study.

OBJECTIVE: To compare the short-term effects and safety of ultrasound (US)-guided sacroiliac joint (SIJ) injections with fluoroscopy (FL)-guided SIJ injections in patients with noninflammatory SIJ dysfunction. DESIGN: Prospective, randomized controlled trial. SETTING: University hospital. PARTICIPANTS: Patients (N=120) with noninflammatory sacroiliac arthritis were enrolled. INTERVENTION: All procedures were performed using an FL or US apparatus. Subjects were randomly assigned to either the FL or US group. Immediately after the SIJ injections, fluoroscopy was applied to verify the correct placement of the injected medication and intravascular injections. MAIN OUTCOME MEASURES: Treatment effects and functional improvement were compared at 2 and 12 weeks after the procedures. RESULTS: The verbal numeric pain scale and Oswestry Disability Index improved at 2 and 12 weeks after the injections without statistical significances between groups. Of 55 US-guided injections, 48 (87.3%) were successful and 7 (12.7%) were missed. The FL-guided SIJ approach exhibited a greater accuracy (98.2%) than the US-guided approach. Vascularization around the SIJ was seen in 34 of 55 patients. Among the 34 patients, 7 had vascularization inside the joint, 23 had vascularization around the joint, and 4 had vascularization both inside and around the joint. Three cases of intravascular injections occurred in the FL group. CONCLUSIONS: The US-guided approach may facilitate the identification and avoidance of the critical vessels around or within the SIJ. Function and pain relief significantly improved in both groups without significant differences between groups. The US-guided approach was shown to be as effective as the FL-guided approach in treatment effects. However, diagnostic application in the SIJ may be limited because of the significantly lower accuracy rate (87.3%).

Intrathecal Baclofen Injection Efficacy for Spasticity Management in Patients With Stroke: A Meta-Analysis.

Although intrathecal baclofen injections have been used for spasticity management regarding stroke, spinal cord injury, and central nervous system diseases, their relative efficacy is controversial. This systematic review scoured 3 multinational electronic databases (Cochrane Library, MEDLINE, and Embase) to isolate relevant studies. We analyzed non-randomized studies and randomized control trials (RCTs) with direct comparisons against other spasticity management interventions for adult stroke patients. Risk of Bias (RoB) and the Risk of Bias Assessment tool for Non-randomized Studies evaluations were implemented with Cochrane's RoB tool. Meta-analysis was performed with Revman 5.4, and evidence validity was assessed with the Grading of Recommendations, Assessment, Development, and Evaluations method. Lastly, the intrathecal baclofen injection meta-analysis included 2 RCTs and 7 non-RCTs for assessing spasticity and 4 non-RCTs to measure gait velocity. Based on this data, intrathecal baclofen injection significantly impacted spasticity and gait speed. Thus, intrathecal baclofen injection can potentially treat severe spasticity unresponsive to conventional spasticity therapy. Furthermore, clinicians must consider individual patient characteristics and conditions when contemplating intrathecal baclofen injection for spasticity intervention.

Treatment effects of ultrasound-guided capsular distension with hyaluronic acid in adhesive capsulitis of the shoulder.

OBJECTIVE: To investigate the efficacy of ultrasound-guided intra-articular (IA) hyaluronic acid injection with capsular distension compared with steroid injection alone in patients with adhesive capsulitis of the shoulder by assessing pain relief, functional improvements, and range of motion at 2 and 6 weeks after final injections. DESIGN: Prospective randomized controlled trial. SETTING: University hospital. PARTICIPANTS: Patients (N=100) with adhesive capsulitis of shoulder. INTERVENTIONS: Subjects were randomly assigned to 2 groups: 45 patients in group A were treated with 0.5% lidocaine plus triamcinolone 40mg IA injection and 45 patients in group B were treated with 0.5% lidocaine plus hyaluronic acid 20mg and capsular distension. All injections were performed every 2 weeks for a total of 3 times. MAIN OUTCOME MEASURES: Treatment effects were assessed using the Shoulder Pain and Disability Index (SPADI), Verbal Numeric Scale (VNS), and passive range of motion (ROM) of the shoulder (flexion, abduction, external rotation) before injections and at 2 and 6 weeks after the last injections. RESULTS: SPADI, VNS, and passive ROM were improved at 2 and 6 weeks in both groups. The statistical differences were not observed in SPADI and VNS between groups (P<.05), and shoulder passive external rotation was more improved in group B than in group A (P<.05). CONCLUSIONS: Capsular distension with IA hyaluronic acid injection was shown to be a treatment method as effective as the steroid injection alone in pain relief and functional improvement; additionally, it was more effective in passive external rotation improvement than steroid injection alone.

Ultrasound-guided selective nerve root block versus fluoroscopy-guided transforaminal block for the treatment of radicular pain in the lower cervical spine: a randomized, blinded, controlled study.

OBJECTIVES: To compare the short-term effects and advantages of ultrasound-guided selective nerve root block with fluoroscopy-guided transforaminal epidural block for radicular pain in the lower cervical spine through assessment of pain relief, functional improvement, and safety. METHODS: A total of 120 patients with radicular pain from cervical spinal stenosis or cervical herniated disc were enrolled. All procedures were performed using a fluoroscopy or ultrasound apparatus. The subjects were randomly assigned to either the fluoroscopy (FL) or ultrasound (US) group. The complication frequencies during the procedures, treatment effects, and functional improvement of the nerve root block were compared at 2 and 12 weeks after the procedures. RESULTS: Verbal Numeric Pain Scale (VNS) improved 2 weeks and 12 weeks after the injections in both groups. Statistical differences were not observed in VNS, Neck Disability Index (NDI), and effectiveness between the groups. In 21 patients at US, vessels were identified at the anterior aspect of the foramen. Eleven patients had a critical vessel at the posterior aspect of the foramen and five patients had on artery continue medially into the foramen, forming, or joining a segmental feeder artery. In both cases, the vessels might well have been in the pathway of the needle correctly positioned under fluoroscopic guidance. Five cases of intravascular injections were observed only in FL without significant difference between the groups. CONCLUSIONS: The US-guided method may facilitate identifying critical vessels at unexpected locations relative to the intervertebral foramen and avoiding injury to such vessels, which is the leading cause of the reported complications from cervical transforaminal injections. On treatment effect, using either method of epidural injections to deliver steroids for cervical radicular pain, secondary to herniated intervertebral disc or foraminal stenosis, significant improvements in function and pain relief were observed in both groups after the intervention. However, significant difference was not observed between the groups. Therefore, the ultrasound-guided method was shown to be as effective as the fluoroscopy-guided method in pain relief and functional improvement, in addition to the absence of radiation and avoiding vessel injury at real-time imaging.

Ultrasound versus palpation guidance for intra-articular injections in patients with degenerative osteoarthritis of the elbow.

OBJECTIVES: The aim of this study is to evaluate the accuracy rate of ultrasound (US) -guided intra-articular (IA) injections in patients by posterior approach with osteoarthritis (OA) of the elbow. METHODS: Eighty patients with physical examination and radiographic findings of elbow OA were included. After US-guided IA or palpation-guided IA injection of iohexol contrast by posterior approach into the elbow joint, fluoroscopic images were obtained to ascertain whether the injected material had reached the IA space. RESULTS: Of the 40 injections performed by US guidance, 40 were confirmed to have been placed in the IA space (an accuracy rate of 100%). Thirty-one of the 40 injections performed by the palpation guidance were IA space (77.5% accuracy rate). CONCLUSIONS: US-guided IA elbow injections by posterior approach may raise the accuracy rate in elbow joint injections.

Clinical Practice Guidelines for Oropharyngeal Dysphagia.

OBJECTIVE: Dysphagia is a common clinical condition characterized by difficulty in swallowing. It is sub-classified into oropharyngeal dysphagia, which refers to problems in the mouth and pharynx, and esophageal dysphagia, which refers to problems in the esophageal body and esophagogastric junction. Dysphagia can have a significant negative impact one's physical health and quality of life as its severity increases. Therefore, proper assessment and management of dysphagia are critical for improving swallowing function and preventing complications. Thus a guideline was developed to provide evidence-based recommendations for assessment and management in patients with dysphagia. METHODS: Nineteen key questions on dysphagia were developed. These questions dealt with various aspects of problems related to dysphagia, including assessment, management, and complications. A literature search for relevant articles was conducted using Pubmed, Embase, the Cochrane Library, and one domestic database of KoreaMed, until April 2021. The level of evidence and recommendation grade were established according to the Grading of Recommendation Assessment, Development and Evaluation methodology. RESULTS: Early screening and assessment of videofluoroscopic swallowing were recommended for assessing the presence of dysphagia. Therapeutic methods, such as tongue and pharyngeal muscle strengthening exercises and neuromuscular electrical stimulation with swallowing therapy, were effective in improving swallowing function and quality of life in patients with dysphagia. Nutritional intervention and an oral care program were also recommended. CONCLUSION: This guideline presents recommendations for the assessment and management of patients with oropharyngeal dysphagia, including rehabilitative strategies.

Clinical Practice Guideline for Stroke Rehabilitation in Korea-Part 1: Rehabilitation for Motor Function (2022).

This clinical practice guideline (CPG) is the fourth edition of the Korean guideline for stroke rehabilitation, which was last updated in 2016. The development approach has been changed from a consensus-based approach to an evidence-based approach using the Grading of Recommendations Assessment Development and Evaluation (GRADE) method. This change ensures that the guidelines are based on the latest and strongest evidence available. The aim is to provide the most accurate and effective guidance to stroke rehabilitation teams, and to improve the outcomes for stroke patients in Korea. Fifty-five specialists in stroke rehabilitation and one CPG development methodology expert participated in this development. The scope of the previous clinical guidelines was very extensive, making it difficult to revise at once. Therefore, it was decided that the scope of this revised CPG would be limited to Part 1: Rehabilitation for Motor Function. The key questions were selected by considering the preferences of the target population and referring to foreign guidelines for stroke rehabilitation, and the recommendations were completed through systematic literature review and the GRADE method. The draft recommendations, which were agreed upon through an official consensus process, were refined after evaluation by a public hearing and external expert evaluation.

Usefulness of combined handheld ultrasound and fluoroscopy-guided injection in adhesive capsulitis of the shoulder: A prospective, randomized single blind-pilot study.

BACKGROUND: Ultrasound is increasingly being utilized in the diagnosis and treatment of adhesive capsulitis. OBJECTIVE: To compare the therapeutic effects and advantages of combined handheld ultrasound and fluoroscopy-guided intra-articular corticosteroid injection with those of conventional ultrasound-guided corticosteroid injection in adhesive capsulitis of the shoulder. METHODS: A total of 39 patients diagnosed with adhesive capsulitis of the shoulder were randomly assigned into two groups. Group A patients (n= 19) underwent combined handheld ultrasound and fluoroscopy-guided corticosteroid injection and group B patients (n= 20) underwent conventional ultrasound-guided corticosteroid injection to the intra-articular space of the shoulder twice. Treatment efficacy was assessed at 2 and 6 weeks after the final injection, based on the verbal numeric pain scale, Shoulder Pain and Disability Index, and range of motion. Secondary outcome measures were the accuracy and procedure time. RESULTS: Both injection methods were effective in the treatment of adhesive capsulitis. No significant differences in treatment efficacy and injection accuracy were observed between the two groups (p> 0.05). CONCLUSIONS: This study showed no statistical differences in treatment efficacy between 2 groups. However, the combined use of ultrasound and fluoroscopy can increase the accuracy of injection compared with conventional ultrasound alone.

Effect of Intake of Leucine-Rich Protein Supplement in Parallel with Resistance Exercise on the Body Composition and Function of Healthy Adults.

Although sarcopenia has been dealt with in several studies, the standardized guidelines for preventing sarcopenia resulting from increased life expectancy are still insufficient. Therefore, this study evaluated the effects of daily resistance exercise and the intake of leucine-rich protein supplements daily for 12 weeks on the body composition and physical function of healthy adults aged >50 years living in Korea. The study analyzed 50 healthy people without medical conditions, who were randomly assigned to two groups (taking either protein powder or placebo powder) twice a day for 12 weeks. All participants performed resistance exercises regularly that could be repeated 8−12 times using a TheraBand for 12 weeks. A total of 41 participants completed the study. When measured via bioimpedance analysis (BIA), body fat mass (kg) and body fat (%) significantly decreased, and lean body mass (LBM) (kg) and skeletal muscle mass (SMM) (kg) significantly increased, in both groups. However, when measured via dual-energy X-ray absorptiometry (DXA), LBM was significantly increased only in the protein powder group. The LBM and SMM change measured via BIA was significantly greater in the protein powder group than in the placebo powder group (LBM: 0.95 ± 0.91 kg in the protein powder group vs. 0.38 ± 1.06 kg in the placebo powder group, p = 0.043; SMM: 0.69 ± 0.58 kg in the protein powder group vs. 0.29 ± 0.65 kg in the placebo powder group, p = 0.039, respectively). In the senior fitness test (SFT), significant functional improvement was found within the two groups, but no significant difference was found between the groups in the degree of improvement. In conclusion, in older people aged >50, to prevent sarcopenia, is more effective to combine resistance exercise and leucine-rich protein supplementation than to simply perform resistance exercise.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

A Novel Statin Compound from Monacolin J Produced Using CYP102A1-Catalyzed Regioselective C-Hydroxylation.

Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), which is the rate-limiting enzyme in cholesterol biosynthesis. Statin therapy reduces morbidity and mortality in those who are at high risk of cardiovascular disease. Monacolin J is a statin compound, which is an intermediate in the lovastatin biosynthesis pathway, in the fungus Aspergillus terreus. It is also found in red yeast rice, which is made by culturing rice with the yeast Monascus purpureus. Monacolin J has a hydroxyl substituent at position C'-8 of monacolin L. Here, a new statin derivative from monacolin J was made through the catalysis of CYP102A1 from Bacillus megaterium. A set of CYP102A1 mutants of monacolin J hydroxylation with high catalytic activity was screened. The major hydroxylated product was C-6'a-hydroxymethyl monacolin J, whose structure was confirmed using LC-MS and NMR analysis. The C-6'a-hydroxymethyl monacolin J has never been reported before. It showed a greater ability to inhibit HMG-CoA reductase than the monacolin J substrate itself. Human liver microsomes and human CYP3A4 also showed the ability to catalyze monacolin J in producing the same product of the CYP102A1-catalyzed reaction. This result motivates a new strategy for the development of a lead for the enzymatic and chemical processes to develop statin drug candidates.