Exploring the Higher Order Structure and Conformational Transitions in Insulin Microcrystalline Biopharmaceuticals by Proton-Detected Solid-State Nuclear Magnetic Resonance at Natural Abundance.

The integrity of a higher order structure (HOS) is an essential requirement to ensure the efficacy, stability, and safety of protein therapeutics. Solution-state nuclear magnetic resonance (NMR) occupies a unique niche as one of the most promising methods to access atomic-level structural information on soluble biopharmaceutical formulations. Another major class of drugs is poorly soluble, such as microcrystalline suspensions, which poses significant challenges for the characterization of the active ingredient in its native state. Here, we have demonstrated a solid-state NMR method for HOS characterization of biopharmaceutical suspensions employing a selective excitation scheme under fast magic angle spinning (MAS). The applicability of the method is shown on commercial insulin suspensions at natural isotopic abundance. Selective excitation aided with proton detection and non-uniform sampling (NUS) provides improved sensitivity and resolution. The enhanced resolution enabled us to demonstrate the first experimental evidence of a phenol-escaping pathway in insulin, leading to conformational transitions to different hexameric states. This approach has the potential to serve as a valuable means for meticulously examining microcrystalline biopharmaceutical suspensions, which was previously not attainable in their native formulation states and can be seamlessly extended to other classes of biopharmaceuticals such as mAbs and other microcrystalline proteins.

Combined Liquid-State and Solid-State Nuclear Magnetic Resonance at Natural Abundance for Comparative Higher Order Structure Assessment in the Formulated-State of Biphasic Biopharmaceutics.

A higher-order structure (HOS) is critical to a biopharmaceutical drug as the three-dimensional structure governs its function. Even the partial perturbation in the HOS of the drug can alter the biological efficiency and efficacy. Due to current limitations in analytical technologies, it is imperative to develop a protocol to characterize the HOS of biopharmaceuticals in the native formulated state. This becomes even more challenging for the suspension formulations where solution and solid phases co-exist. Here, we have used a combinatorial approach using liquid (1D 1H) and solid-state (13C CP MAS) NMR methodology to demonstrate the HOS in the biphasic microcrystalline suspension drug in its formulated state. The data were further assessed by principal component analysis and Mahalanobis distance (DM) calculation for quantitative assessment. This approach is sufficient to provide information regarding the protein HOS and the local dynamics of the molecule when combined with orthogonal techniques such as X-ray scattering. Our method can be an elegant tool to investigate batch-to-batch variation in the process of manufacture and storage as well as a biosimilarity comparison study for biphasic/microcrystalline suspension.

Outcome of re-operation for local recurrence following pre-operative denosumab administration and curettage for giant cell tumour of bone with difficult joint preservation.

PURPOSE: Denosumab enables joint-sparing surgery (curettage) and surgical downstaging in patients with giant cell tumour of bone (GCTB), where joint preservation is not possible. However, denosumab treatment causes osteosclerosis of the lesion, making it difficult to curet the lesion, leaving the tumour behind, and increasing the local recurrence rate. We performed a three-centre retrospective study to investigate the postoperative local re-recurrence rate, joint preservation status, and functional outcomes of locally recurrent lesions after preoperative denosumab treatment and curettage in patients with difficult joint preservation. METHODS: We included 38 of 142 patients with primary GCTB of the extremities who underwent preoperative denosumab and curettage between 2009 and 2021 with local recurrence. Preoperative denosumab was indicated in patients with minimal residual periarticular and subchondral bones, large extraosseous lesions (Campanacci stage 3), and pathological fractures that made joint preservation difficult. RESULTS: Local re-recurrence occurred in 6 (15.8%) of the 38 patients. In 29 patients who underwent re-curettage, local re-recurrence occurred in six patients (20.7%); however, in nine patients who underwent en bloc resection, no local re-recurrence was observed. The joint preservation rate was 63.2% (24 of 38 patients), with a median Musculoskeletal Tumor Society score of 28 (interquartile range: 26.8-29.0). The median follow-up period after surgery for local recurrence was 63.5 months (interquartile range: 42.5-82.4). CONCLUSION: Since the local re-recurrence rate after re-curettage for local recurrence was low, and the joint preservation rate and affected limb function were good, preoperative denosumab administration may be considered in patients who require downstaging to maintain good limb function (joint preservation).

Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks.

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

Nogo receptor decoy promotes recovery and corticospinal growth in non-human primate spinal cord injury.

After CNS trauma such as spinal cord injury, the ability of surviving neural elements to sprout axons, reorganize neural networks and support recovery of function is severely restricted, contributing to chronic neurological deficits. Among limitations on neural recovery are myelin-associated inhibitors functioning as ligands for neuronal Nogo receptor 1 (NgR1). A soluble decoy (NgR1-Fc, AXER-204) blocks these ligands and provides a means to promote recovery of function in multiple preclinical rodent models of spinal cord injury. However, the safety and efficacy of this reagent in non-human primate spinal cord injury and its toxicological profile have not been described. Here, we provide evidence that chronic intrathecal and intravenous administration of NgR1-Fc to cynomolgus monkey and to rat are without evident toxicity at doses of 20 mg and greater every other day (≥2.0 mg/kg/day), and far greater than the projected human dose. Adult female African green monkeys underwent right C5/6 lateral hemisection with evidence of persistent disuse of the right forelimb during feeding and right hindlimb during locomotion. At 1 month post-injury, the animals were randomized to treatment with vehicle (n = 6) or 0.10-0.17 mg/kg/day of NgR1-Fc (n = 8) delivered via intrathecal lumbar catheter and osmotic minipump for 4 months. One animal was removed from the study because of surgical complications of the catheter, but no treatment-related adverse events were noted in either group. Animal behaviour was evaluated at 6-7 months post-injury, i.e. 1-2 months after treatment cessation. The use of the impaired forelimb during spontaneous feeding and the impaired hindlimb during locomotion were both significantly greater in the treatment group. Tissue collected at 7-12 months post-injury showed no significant differences in lesion size, fibrotic scar, gliosis or neuroinflammation between groups. Serotoninergic raphespinal fibres below the lesion showed no deficit, with equal density on the lesioned and intact side below the level of the injury in both groups. Corticospinal axons traced from biotin-dextran-amine injections in the left motor cortex were equally labelled across groups and reduced caudal to the injury. The NgR1-Fc group tissue exhibited a significant 2-3-fold increased corticospinal axon density in the cervical cord below the level of the injury relative to the vehicle group. The data show that NgR1-Fc does not have preclinical toxicological issues in healthy animals or safety concerns in spinal cord injury animals. Thus, it presents as a potential therapeutic for spinal cord injury with evidence for behavioural improvement and growth of injured pathways in non-human primate spinal cord injury.

Hevea brasiliensis coniferaldehyde-5-hydroxylase (HbCAld5H) regulates xylogenesis, structure and lignin chemistry of xylem cell wall in Nicotiana tabacum.

KEY MESSAGE: The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl-guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency.

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

A Comparison of Outcomes After Endoscopic Repair of Partial- Versus Full-Thickness Tears of the Gluteus Medius Tendon.

PURPOSE: The purpose of this study was to evaluate and compare the functional outcomes after endoscopic repair of partial or full-thickness gluteus medius tears at a minimum 2 years' follow-up. METHODS: Patients with isolated tears of the gluteus medius tendon repaired endoscopically between 2012 and 2017 were evaluated at a minimum 2 years of follow-up. Patients with large, retracted and/or irreducible tears, advanced atrophy, or fatty degeneration or with concomitant other hip pathology were excluded. Functional outcomes were assessed using the modified Harris Hip Score (mHHS), Nonarthritic Hip Score (NAHS), and visual analog scale (VAS) for pain preoperatively and at the last follow-up RESULTS: Forty-six patients, 3 men and 43 women with a mean (SD) age of 63 (9; range, 43-82) years, were included. Thirty-three patients (72%) had partial-thickness tears and 13 (28%) had full-thickness tears. Pain was reduced significantly from a median of 8 (IQR: 6-8) at the preoperative visit to a median of 2 (IQR: 15) at the most recent follow-up (P < .0001). The mHHS and NAHS improved significantly from a median of 44 (IQR: 35-52) to 80 (IQR: 64-87) (P < .0001) and 52 (IQR: 43-56) to 75 (IQR: 66-94) (P < .0001), respectively. These improvements were clinically relevant and surpass the reported minimal clinically important difference. Patients with partial tears had significant functional gains in the mHHS in comparison with patients with full-thickness tears (P = .02). No other statistically significant difference between groups of tear grade was observed. CONCLUSIONS: Endoscopic repair of gluteus medius tendon tears yields excellent functional outcomes at a minimum follow-up of 2 years. A lower functional improvement for full-thickness tears was observed; this difference was statistically significant for 1 of the 2 scores evaluated. LEVEL OF EVIDENCE: Level III, retrospective comparative cohort.

Safety Evaluation of KP-10 (Metastin 45-54) Following once Daily Intravenous Administration for 14 Days in Dog.

Kisspeptin-10 (previously referred as metastin 45-54), an active fragment of the endogenous full-length kisspeptin-145, is a potential therapeutic agent for reproductive disorders such as infertility, amenorrhea, and pubertal delay. A safety evaluation of KP-10 was conducted in dogs at the doses of 30, 100, and 1,000 µg/kg, given once daily intravenously for 14 days with a 14-day recovery period. There were no overt signs of drug-related toxicity observed in clinical signs, body weights, food consumption, clinical pathology, histopathology, urinalysis, electrocardiogram, or respiratory rate. Due to very rapid clearance of the peptide, luteinizing hormone (LH) levels were measured as a surrogate marker to demonstrate KP-10 exposure. The LH response reached a maximum concentration at 5 minutes post-dose and remained relatively unchanged for at least 30 minutes after dosing with no gender effect. LH concentrations on Day 1 were generally greater than on day 14. Vaginal cytology results indicated all dogs were in anestrous throughout the dosing period. There were also no KP-10-related findings observed in recovery animals on Day 29. In conclusion, KP-10 demonstrated favorable safety profile in dog where 1,000 µg/kg dose was considered as a no-observed-adverse-effect level dose when administered IV once daily for 14 days.

Safety Assessment of AAV2-hGDNF Administered Via Intracerebral Injection in Rats for Treatment of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective biologic in Parkinson's disease models. Adeno-associated viral vector serotype 2 (AAV2)-human GDNF safety was assessed in rats treated with a single intracerebral dose of vehicle, 6.8 × 108, 6.8 × 109, or 5.2 × 1010 vector genomes (vg)/dose followed by interim sacrifices on day 7, 31, 90, and 376. There were no treatment-related effects observed on food consumption, body weight, hematology, clinical chemistry, coagulation parameters, neurobehavioral parameters, organ weights, or serum GDNF and anti-GDNF antibody levels. Increased serum anti-AAV2 neutralizing antibody titers were observed in the 5.2 × 1010 vg/dose group. Histopathological lesions were observed at the injection site in the 6.8 × 109 vg/dose (day 7) and 5.2 × 1010 vg/dose groups (days 7 and 31) and consisted of gliosis, mononuclear perivascular cuffing, intranuclear inclusion bodies, and/or apoptosis on day 7 and mononuclear perivascular cuffing on day 31. GDNF immunostaining was observed in the injection site in all dose groups through day 376 indicating no detectable impacts of anti-AAV2 neutralizing antibody. There was no evidence of increased expression of calcitonin gene-related peptide or Swann cell hyperplasia in the cervical and lumbar spinal cord or medulla oblongata at the 5.2 × 1010 vg/dose level indicating lack of hyperplastic effects. In conclusion, no systemic toxicity was observed, and the local toxicity observed at the injection site appeared to be reversible demonstrating a promising safety profile of intracerebral AAV2-GDNF delivery. Furthermore, an intracerebral dose of 6.8 × 108 AAV2-GDNF vg/dose was considered to be a no observed adverse effect level in rats.

Posterolateral approach for all-inside arthroscopic lateral meniscus repair in athletes: technique and outcomes.

INTRODUCTION: The past 2 decades have been marked by substantial progress in our knowledge of meniscus anatomy, function, and biomechanics, and also by the shifting of the surgical treatment of meniscal lesions from traditional meniscectomy towards arthroscopic repair to get away from the early osteoarthritis associated with meniscectomy. Posterior horn injuries of the lateral meniscus (LM) have been less studied due to their lower incidence and also due to the historical technical complexity of performing a repair in the posterolateral compartment. MATERIALS AND METHODS: A retrospective analysis of prospectively collected data was performed of all athletic patients who had a peripheral longitudinal tear of the lateral meniscal posterior horn and who underwent at least one repair procedure with a posterolateral approach between 2014 and 2018. The type of injury, extent of lateral meniscal tear, and characteristics of sutures placed were assessed. Clinical assessment included objective and subjective IKDC ratings. The Tegner activity level score was determined before the injury and at the last follow-up visit. Failure was defined as a need for revision surgery. All complications were documented. RESULTS: The study population comprised 24 athletes with a mean follow-up of 25.2 ± 10 months. The Tegner activity level was exactly the same before the injury as after the surgery. The mean IKDC score significantly increased from 41.8 (12.2) before the surgery to 94.5 (9.1) after. There were four reoperations for failure (16.6%) that required a new suture repair. None of these revised repairs sustained a new failure as of the last follow-up. CONCLUSION: Despite the long learning curve, the posterolateral approach is a safe and effective technique for longitudinal tears of the posterior horn of the LM. The results of all-inside suture repair through a posterolateral portal are comparable to other techniques.

Design and synthesis of new indanol-1,2,3-triazole derivatives as potent antitubercular and antimicrobial agents.

In a search of new antitubercular agents, herein we have reported a series of new thirty-two indanol-1,2,3-triazole derivatives. The synthesized compounds were screened for their in vitro antitubercular and antimicrobial activities. Among the screened compounds, most of the compounds have displayed good antitubercular activity against Mycobacterium tuberculosis H37Rv. The compound 5g has been identified as potent antitubercular agent with MIC value 1.56 µM. The most active compounds of the series were further studied for their cytotoxicity against HEK 293 cells using MTT assay and found to be nontoxic. In addition, ten compounds were shown good antimicrobial activities against both antibacterial and antifungal pathogens. A molecular docking study against Mycobacterial enoyl-ACP-reductase (InhA) was performed to gain an insight into the molecular mechanism of antitubercular action. The pharmacokinetic parameters of these compounds were studied and displayed acceptable drug-likeness score.

Safety assessment of metarrestin in dogs: A clinical candidate targeting a subnuclear structure unique to metastatic cancer cells.

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median Tmax) across all doses with a mean t1/2 over 168 h of 55.5 h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmax = 82.5 ng/mL and AUClast = 2521 h*ng/mL, on Day 27.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks.

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

What Are the Challenges and Complications of Sterilizing Autografts with Liquid Nitrogen for Malignant Bone Tumors? A Preliminary Report.

BACKGROUND: Reconstruction of defects after resection of malignant bone tumors with liquid nitrogen-sterilized recycled autografts is an alternative to bone allografts and endoprostheses in resource-constrained environments. Most studies reporting favorable outcomes with liquid nitrogen-sterilized autografts for bone reconstruction are geographically restricted to a few countries, and the technical challenges of routinely using liquid nitrogen intraoperatively, especially when using the pedicle freezing technique, has not been documented. QUESTIONS/PURPOSES: (1) What are the technical challenges of liquid nitrogen sterilization of bone tumors for inexperienced surgeons? (2) What are the complications associated with the procedure? METHODS: Between May 2017 and October 2019, 88 patients underwent limb salvage procedures for malignant bone tumors of the extremities at our institution. An endoprosthesis was used for reconstruction of the defect following resection in 45% (40 of 88) of these patients, mostly in adults (median age 21 years; range 9 to 68). In the remaining 55% (48 of 88) of patients undergoing biological reconstruction, liquid nitrogen-sterilized autograft was used in 90% (43 of 48), extracorporeal irradiation-sterilized autograft was used in 4% (2 of 48) and allograft was used in 6% (3 of 48). Of the 43 patients receiving liquid nitrogen-sterilized autograft, 5% (2 of 43) were excluded due to loss to follow-up and the remaining 95% (41 of 43) were included for the analysis. Liquid nitrogen-sterilized autograft was the preferred method of reconstruction at our institution during the study period, unless the patient had an indication for prosthesis reconstruction; extracorporeal irradiation-sterilized autograft was used due to resource constraints with liquid nitrogen and allograft was used when patients insisted.All surgical procedures were performed by the same team of trained orthopaedic oncology surgeons. The medical records of the included 41 patients were retrieved using an institutional database search in this retrospective study, and all were used to ascertain technical challenges associated with the operations as well as early (within 3 weeks of the index procedure) and late complications (those occurring 3 weeks or more after surgery). The technical challenges were defined as follows: the quantity of liquid nitrogen to be used; arranging, storing and handling of liquid nitrogen in the operating room, type and size of the container to be used for sterilization, the positioning of the container during pedicle freezing, level of fibular osteotomy for pedicle freezing of tibia, soft tissue protection, limb rotation during pedicle freezing, managing tourniquet time, and any other intraoperative factors with the use of liquid nitrogen for sterilizing the autograft. As our experience with the technique gradually grew, the answers to the above-mentioned factors were determined. Considering the removal of autograft as the endpoint of interest, survival of the autograft was determined by Kaplan-Meier analysis.The median (range) patient age was 14 years (2 to 49), and 54% (22 of 41) were males. Osteosarcoma was the most common diagnosis (68%, [28 of 41]) followed by Ewing's sarcoma (20%, [8 of 41]). On presentation, 27% of patients (11 of 41) had radiological evidence of pulmonary metastasis. Tumors were seen frequently around the knee (39% [16 of 41] proximal tibia and 22% [9 of 41] distal femur). Before resection 85% (35 of 41) underwent neoadjuvant chemotherapy. Sixty-six percent (27 of 41) underwent pedicle-freezing and the remaining 34% (14 of 41) underwent free-freezing of the tumor segment of the bone. The median (range) duration of surgery was 280 minutes (210 to 510). The patients were followed up for a median (range) duration of 21 months (5 to 30); two patients were lost to follow-up. RESULTS: With gradual experience using liquid nitrogen-sterilization over time at our institution, we determined that the following factors helped us in performing liquid nitrogen-sterilization more efficiently. For every operation 15 L to 20 L of unsterilized liquid nitrogen was arranged, 1 or 2 days before the procedure, and stored in industrial-grade cryocylinders in the operating complex. During the procedure, the operating surgeons wore additional plastic aprons under the surgical gowns, surgical goggles, and rubber boots. The staff managing the liquid nitrogen in the operating room wore thermal protective gloves. For most of the pedicle freezing procedures, we used a cylindrical stainless-steel container that was 30 cm in height and 15 cm in diameter, with a narrow opening. The container was kept on a separate moveable cart that was placed next to the operating table at a slightly lower level, and it was wrapped in multiple cotton rolls, plastic sheets, surgical sheets, and a crepe bandage. For pedicle freezing of the tibia, we performed the fibular osteotomy at least 5 cm away from the planned surgical margin, roughly around the axis of rotation of the limb. The soft tissue at the base of the delivered bone segment was dissected for at least 5 cm beyond the planned surgical margin of bone, and was protected with multiple layers of cotton rolls, plastic drapes, a single roll of Esmarch and crepe bandage. The tumor segment was externally rotated during pedicle freezing for all anatomic sites (proximal tibia, distal tibia, proximal humerus, and proximal femur). The tourniquet was inflated just before pedicle freezing to prevent tumor dissemination and not before the initial incision in all pedicle freezing procedures.Thirty-nine percent of patients (16 of 41) experienced complications associated with the procedures, and 15% (6 of 41) underwent revision surgery. Early complications (occurring within 3 weeks of the index procedure) were skin necrosis in four of 16 patients, intraoperative fracture in one of 16, superficial infection in one of 16, and neurapraxia in one of 16 patients. Late complications (occurring 3 weeks or more after surgery) were resorption of the recycled bone in four of 16 patients, nonunion of the osteotomy site in two of 12, delayed union of the osteotomy site in one of 16, collapse of the recycled bone in one of 16, and local recurrence in 1 of 16 patients. Kaplan-Meier survivorship free from removal of autograft at 2 years after surgery was 92% (95% confidence interval 89 to 96). CONCLUSION: Liquid nitrogen-sterilization is an alternative technique that requires some training and experience for the surgeon to become proficient in treating primary malignant bone tumors. Because it is widely available, it may be an option worth exploring in resource-constrained environments, where allografts and endoprostheses cannot be procured. The methods we developed to address the technical challenges will require more study and experience, but we believe these observations will aid others who may wish to use and evaluate liquid nitrogen sterilization of extremity bone sarcomas. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Is a Short-course of Preoperative Denosumab as Effective as Prolonged Therapy for Giant Cell Tumor of Bone?

BACKGROUND: Denosumab is an inhibitor of monoclonal receptor activator of nuclear factor-ĸB ligand, approved to treat giant cell tumors of bone (GCTB). It is commonly used for unresectable tumors and for downstaging the tumor to perform less-morbid procedures. Although denosumab has been used extensively for GCTBs, there are no recommendations regarding the duration of therapy. The risk factors associated with local recurrence (LR) in patients receiving preoperative denosumab for GCTB also are unknown. QUESTIONS/PURPOSES: (1) Is short-course (three doses or fewer) preoperative denosumab treatment as effective as longer course (more than three doses) of treatment in terms of achieving a clinical, radiologic, and histologic response in patients with GCTB? (2) Is there an increased risk of LR after short-course denosumab therapy compared with long-course denosumab therapy; and after controlling for confounding variables, what factors were associated with LR after surgery for GCTB in patients receiving preoperative denosumab? METHODS: A retrospective study was performed using an institutional database of 161 skeletally mature patients with a histologic diagnosis of GCTB who received denosumab between November 2010 and July 2019 to downstage the tumor before surgery. In general, we used denosumab when we thought it would facilitate either resection or curettage (by formation of a sclerotic rim around the osteolytic lesion), when a less-morbid procedure than initially planned might be performed, and in patients with complex presentations like cortical breech and soft tissue extension, pathological fracture, thinning of more than three cortices of the extremity. From 2010 to late 2015, denosumab was administered for approximately 4 to 6 months; starting in late 2015 through 2020, the number of denosumab doses has been reduced. We divided patients into two groups: Those who received three or fewer doses of denosumab (short-course, n = 98) and those who received more than three doses of denosumab (long-course, n = 63). Comparing those in the long-course group with those in the short-course group whose procedures were performed at least 2 years ago, there were no differences in loss to follow-up before 2 years (3% [3 of 98] versus. 3% [2 of 63]). The mean patient age was 30 years (± 6.1) and the mean number of denosumab doses was 4.4 (range 1 to 14). Overall, 77% (37 of 48) of patients taking short-course denosumab and 75% (27 of 36) of patients on long-course denosumab underwent curettage, and the remaining patients with an inadequate bony shell around the tumor or destruction of articular cartilage in both groups underwent tumor resection. With the numbers available, the patients with short- and long-course denosumab were not different in terms of age, sex, MSTS score on presentation, lesion size, lesion location, Campanacci grade, presence of pathological fracture and pulmonary metastasis on presentation, and the type of surgery performed (curettage versus resection). We analyzed the change in the Musculoskeletal Tumor Society score, change in Campanacci grade, radiologic objective tumor response (defined as a partial or complete response, per the modified inverse Choi criteria), and histologic response (defined as reduction of more than 90% of osteoclast-like giant cells or a reduction of more than 50% of mesenchymal spindle-like stromal cells, along with evidence of lamellar or woven bone formation, when compared with the biopsy sample) between the two groups (short- and long-course denosumab). LR rates were compared between the two groups, and after controlling for confounding variables, factors associated with LR in all operated patients were analyzed with a Cox proportional hazards regression analysis. RESULTS: With the numbers available, there was no difference between the short- and long-course denosumab groups in terms of mean percentage improvement in MSTS score (20 [± 18.5] versus 24 [± 12.6]; p = 0.37), radiologic objective tumor response (90% [43 of 48] versus 81% [29 of 36]; p = 0.24) and histologic response (79% [38 of 48] versus 83% [30 of 36]; p = 0.81). With the numbers available, there was no difference between the short- and long-course denosumab groups in terms of Kaplan-Meier survivorship free from LR at 5 years after surgery (73% [95% confidence interval, 68 to 76] versus 64% [95% CI 59 to 68]; log-rank p = 0.50). After controlling for potential confounding variables like age, sex, Campanacci grade and MSTS score on presentation, number of denosumab doses administered before surgery, clinical, radiologic and histologic response to denosumab, and time duration between denosumab therapy and surgery, we found that tumors involving the bones of the hand and the foot (hazard ratio 7.4 [95% CI 2.0 to 27.3]; p = 0.009) and curettage (HR 6.4 [95% CI 2.8 to 23.0]; p = 0.037) were independently associated with a higher risk of LR. CONCLUSIONS: In this preliminary, single-center study, we found that a short-course of preoperative denosumab (three or fewer doses) was associated with no differences in clinical scores, histological and radiological response, or LR-free survivorship, compared with longer-course of denosumab (more than three doses). Fewer preoperative doses can reduce the complications and costs associated with more-prolonged therapy. Denosumab must be used cautiously before curettage for GCTB, and only if the benefit of joint salvage outweighs the possibility of LR. However, given the small number of patients, potentially clinically important differences might have been missed, and so our findings need to be confirmed by larger, multicenter, prospective trials. LEVEL OF EVIDENCE: Level III, therapeutic study.

The Canadian Congenital Diaphragmatic Hernia Collaborative Mobile App: A National Guideline Implementation Strategy.

OBJECTIVE: Coinciding with the publication of the Canadian congenital diaphragmatic hernia (CDH) Collaborative's clinical practice guidelines (CPG), we developed a mobile smartphone app to increase guideline utilization and promote knowledge translation. STUDY DESIGN: This mobile app was organized into sections corresponding to the phases of CDH care (prenatal, perinatal/postnatal, and child/adolescent), and contained 22 recommendations supported by evidence summaries, PubMed links, levels of evidence, and strength of expert consensus. Download statistics were collected from September 2018 to June 2020 after release of two iOS versions and an Android platform. Data regarding user numbers/location, most visited sections, and individual session details were analyzed. RESULTS: During the study period, the CDH app had 1,586 users predominantly from Canada (40%), United States (30%), and Brazil (12%). The Android release increased app visibility, particularly in Brazil, which had the largest number of new users. Of 3,723 sessions, roughly one-third were returning users. The average session duration and screens viewed/session was 4 minutes and seven screens, respectively. Postnatal ventilation was the most frequently visited subsection after prenatal diagnosis/risk stratification. Measurement of observed-to-expected lung head ratio was the most visited individual recommendation. The guideline compliance checklist was the most frequently accessed resource highlighting its utility. CONCLUSION: The CDH app is an innovative platform to disseminate guidelines. The increasing global reach of the app suggests worldwide CPG relevance. With additional features planned, the CDH app will continue to support clinical decision-making and empower patients and families as they navigate the short and long-term challenges associated with CDH. KEY POINTS: · Mobile smartphone technology provides an optimal platform for guideline dissemination.. · International uptake supports worldwide CPG relevance.. · Future initiatives include the development of patient and family resources..

Preclinical toxicity evaluation of JD5037, a peripherally restricted CB1 receptor inverse agonist, in rats and dogs for treatment of nonalcoholic steatohepatitis.

JD5037 is a novel peripherally restricted CB1 receptor (CB1R) inverse agonist being developed for the treatment of visceral obesity and its metabolic complications, including nonalcoholic fatty liver disease and dyslipidemia. JD5037 was administered by oral gavage at 10, 40, and 150 mg/kg/day dose levels for up to 34 days to Sprague Dawley rats, and at 5, 20, and 75 mg/kg/day dose levels for 28 consecutive days to Beagle dogs. In rats, higher incidences of stereotypic behaviors were observed in 10 mg/kg females and 40 mg/kg males, and slower responses for reflex and sensory tests were observed only in males at 10 and 40 mg/kg during neurobehavioral testing. Sporadic minimal incidences of decreased activity (males) and seizures (both sexes) were observed in rats during daily clinical observations, without any clear dose-relationship. Male dogs at 75 mg/kg during treatment period, but not recovery period, had an increased incidence of gut associated lymphoid tissue hyperplasia and inflammation in the intestine. In both species, highest dose resulted in lower AUCs indicative of non-linear kinetics. Free access to food increased the plasma AUC∞ by ~4.5-fold at 20 mg/kg in dogs, suggesting presence of food may help in systemic absorption of JD5037 in dogs. Based on the study results, 150 mg/kg/day in rats, and 20 and 75 mg/kg/day doses in male and female dogs, respectively, were determined to be the no-observed-adverse-effect-levels (NOAELs).

Intravenous toxicity and toxicokinetics of an HDL mimetic, Fx-5A peptide complex, in cynomolgus monkeys.

Fx-5A peptide complex (Fx-5A), a High Density Lipoproteins (HDL) mimetic, has been shown to reduce atherosclerosis. The safety and toxicokinetics of Fx-5A administered IV by 30 min infusion at 8, 25 or 75 mg/kg body weight or vehicle, once every other day for 27 days, were assessed in cynomolgus monkeys. The Fx-5A was well tolerated at all doses. At the highest dose, there were statistically significant effects on hematology and clinical chemistry parameters that were considered non-adverse. Dose-dependent recoverable non-adverse erythrocytes morphological changes (acanthocytes, echinocytes, spherocytes, microcytes, and/or schistocytes) were observed. Fx-5A was not hemolytic in in-vitro fresh NHP or human blood assay. There were no Fx-5A-related statistically significant changes for any cardiovascular function, ECG or respiratory parameters, when compared to control. In addition, there were no Fx-5A-related effects on organ weights, macroscopic or microscopic endpoints. Finally, Fx-5A exhibited sporadic non-appreciable detection of anti-Fx-5A antibodies and a dose-dependent linear toxicokinetics with T1/2 value ranges from 2.7 to 6.2 h. In conclusion, the No Observed Adverse Effect Level was considered to be 75 mg/kg/day with associated exposures average Cmax and AUC0-last of 453 µg/mL and 2232 h µg/mL, respectively, on Day 27.

Banana lectin (BanLec) induces non-specific activation of basophils and mast cells in atopic subjects.

Summary: Dietary lectins play a major role in the activation of mast cells / basophils by bridging cell surface IgE glycans to release histamine and other mediators. In the present study, the effect of mannose / glucose-specific banana lectin (BanLec) on the activation of mast cells / basophils from non-atopic and atopic subjects has been investigated. BanLec was purified from banana pulp in a yield of 7 mg/kg. Leukocytes isolated from heparinized blood of non-atopic / atopic subjects were used for quantitation of the released histamine. Approximately 28.2% of the atopics (n = 117) was positive by skin prick test (SPT) to purified BanLec (100 µg/mL concentration), and all the non-atopics (n = 20) were negative. Maximal release of histamine was seen at 2 µg of BanLec. In percent histamine release, an increase of 35-40% is observed in case of atopics (n = 7) compared to non-atopics (n = 5), and the histamine release from atopic and non-atopic subjects correlates fairly well with the total serum IgE levels (R2 = 0.817). BanLec also induces release of histamine (26.7%) from mast cells present in rat peritoneal exudate cells. BanLec can significantly activate and degranulate mast cells and basophils by cross-linking the trimannosidic core mannose of IgE glycans in atopic population as compared to non-atopic population; the activation is marginal in the case of non-atopics.