Formulation and Characterization of Conjugate Vaccines to Reduce Opioid Use Disorders Suitable for Pharmaceutical Manufacturing and Clinical Evaluation.

This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.

Left-right differences in the proximal femur's strength of post-menopausal women: a multicentric finite element study.

UNLABELLED: The strength of both femurs was estimated in 198 post-menopausal women through subject-specific finite element models. Important random differences between contralateral femurs were found in a significant number of subjects, pointing to the usefulness of further studies to understand if strength-based classification of patients at risk of fracture can be affected by laterality issues. INTRODUCTION: Significant, although small, differences exist in mineral density and anatomy of contralateral proximal femurs. These differences, and their combined effect, may result in a side difference in femurs' strength. However, this has never been tested on a large sample of a homogenous population. METHODS: The strength of both femurs was estimated in 198 post-menopausal women through CT-derived finite element models, built using a validated procedure, in sideways fall conditions. The impact of the resulting asymmetry on the classification of subjects at risk of fracture was analysed. RESULTS: The small difference observed between sides (the right femur on average 4 % stronger than the left) was statistically significant but mechanically negligible. In contrast, higher random differences (absolute difference between sides with respect to mean value) were found: on average close to 15 % (compared to 9.2 % for areal bone mineral density (aBMD) alone), with high scatter among the subjects. When using a threshold-based classification, the right and left femurs were discordant up to over 20 % of cases (K always lower than 0.60) but the left femur was concordant (mean K = 0.84) with the minimum strength between right and left. CONCLUSION: Considering both femurs may be important when trying to classify subjects at risk of failure with strength estimates. Future studies including fracture assessment would be necessary to quantify the real impact.

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

A comparison between micro-CT and histology for the evaluation of cortical bone: effect of polymethylmethacrylate embedding on structural parameters.

Cortical bone microstructure is an important parameter in the evaluation of bone strength. The aim of this study was to validate the characterization of human cortical bone microarchitecture using microcomputed tomography. In order to do this, microcomputed tomography structural measurements were compared with those obtained through histological examination (the gold standard). Moreover, to calculate structural parameters, microcomputed tomography images have to be binarized with the separation between bone and nonbone structures throughout a global thresholding. As the effect of the surrounding medium on the threshold value is not clear, an easy procedure to find the global uniform threshold for a given acquisition condition is applied. This work also compared the structural parameters of microcomputed tomography cortical sample scan in air or embedded in polymethylmethacrylate; histology was used as a reference. For each acquisition condition, a fixed threshold value was found and was applied on the corresponding microcomputed tomography image for the parameters assessment. Twenty cortical bone samples were collected from human femur and tibia diaphyses. All samples were microcomputed tomography scanned in air, embedded in polymethylmethacrylate, rescanned by microcomputed tomography, examined by histology and finally compared. A good correspondence between the microcomputed tomography images and the histological sections was found. Paired comparisons in cortical porosity, Haversian canal diameter and Haversian canal separation between histological sections and microcomputed tomography cross sections, first in air and then embedded in PolyMethylMethAcrylate, were made: no significant differences were found. None of the comparisons showed significant differences for cortical porosity, Haversian canal diameter and Haversian separation over a three-dimensional volume of interest, between microcomputed tomography scans in air and with samples embedded in PolyMethylMethAcrylate. The very good correlation between bone structural measures obtained from microcomputed tomography datasets and from two-dimensional histological sections confirms that microcomputed tomography may be an efficient tool for the characterization of cortical bone microstructure. Moreover, when the corresponding threshold value for each condition is used, structural parameters determined by microcomputed tomography are not affected by the surrounding medium (PolyMethylMethAcrylate).

Pharmacological mechanisms underlying the efficacy of antibodies generated by a vaccine to treat oxycodone use disorder.

Therapeutic vaccines offer a viable strategy to treat opioid use disorders (OUD) complementary to current pharmacotherapies. The candidate Oxy(Gly)4-sKLH vaccine targeting oxycodone displayed pre-clinical proof of efficacy, selectivity and safety, and it is now undergoing clinical evaluation. To further support its implementation in the clinic, this study tested critical in vivo neuropsychopharmacological properties of the Oxy(Gly)4-sKLH vaccine in rats. While repeated immunizations with Oxy(Gly)4-sKLH were necessary to maintain the antibody response overtime, exposure to free oxycodone did not boost oxycodone-specific antibody levels in vaccinated rats, limiting concerns of immune-related side effects. Immunization with Oxy(Gly)4-sKLH achieved sustained antibody titers over a period of five months following initial vaccination, supporting its potential for providing long-lasting protection. In vivo studies of selectivity showed that vaccination prevented oxycodone-induced but not methadone-induced antinociception, while still preserving the opioid antagonist naloxone's pharmacological effects. Vaccination did not interfere with fentanyl-induced antinociception or fentanyl distribution to the brain. These in vivo data confirm the previously reported in vitro selectivity profile of Oxy(Gly)4-sKLH. Vaccination extended oxycodone's half-life up to 25 h compared to control. While vaccination reduced the reinforcing efficacy of oxycodone in an intravenous self-administration model, signs of toxicity were not observed. These rodent studies confirm that active immunization with Oxy(Gly)4-sKLH induces highly specific and long-lasting antibodies which are effective in decreasing the reinforcing effects of oxycodone while preserving the efficacy of medications used to treat OUD and overdose.

Quality control protocol for in vitro micro-computed tomography.

The aim of this work was to present and discuss a quality control protocol for in vitro micro-computed tomography (microCT), based on the adaptation of the quality control protocols for medical computed tomography. The importance of establishing a quality control protocol is related to the opportunity to identify problems on time comparing the microCT images acquired in different time points, and in this way to verify the performance of the device. The proposed quality control protocol was applied for a long-time monitoring period to verify the stability of the micro-tomographic system over time. The protocol proposed in this study was applied to the histomorphometric characterization of bone tissue, but it can be used on a wide range of in vitro microCT applications. Noise and uniformity tests, taken and adapted to micro-tomographic system by medical standard guidelines of quality control, were performed by the use of a water phantom. An accuracy test was designed and performed by the use of a morphometric calibrated phantom. All these tests were performed during a long-time monitoring period to control the stability of the system. Specific control charts and monitoring parameters for each test were used to represent the monthly measures collected during 20 months and an out of control condition was defined. The reference values (baseline), calculated to control the stability of micro-tomographic system over time, were calculated during acceptance/status test. During the period, no out of control conditions in noise, uniformity and accuracy tests were recorded. However, a changing condition was found in noise test, as showed by using statistical C (P < 0.01) and Kruskal-Wallis (P < 0.05) tests. In particular, a Wilcoxon rank sum test with Bonferroni correction (P < 0.0125) was applied in noise test to investigate which of the comparisons among first five acquisitions of year 2004 (group B.L.) and each group was significant (P < 0.0125). The noise showed a slight but significant increase over the years compared to baseline value; however, no out of control conditions were recorded. Nonetheless, a maintenance service to control the performance of mechanical components of microCT was required and performed.

Dependence of mechanical compressive strength on local variations in microarchitecture in cancellous bone of proximal human femur.

Human cancellous bone is a heterogeneous material. Despite this, most of the published studies report correlations between mechanical properties and morphometric parameters averaged on the whole specimen. This work investigated whether local variations in morphometric parameters were linked to the localized failure regions of cancellous bone. Additionally, it was examined whether local values of morphometric parameters can predict the ultimate stress better than the average bone volume fraction (BV/TV). Cylindrical cancellous bone specimens extracted along the primary compressive group of human femoral heads were studied. These were microCT-imaged to assess the morphometric parameters, compressed to determine the ultimate stress, and rescanned by microCT to visualize the failure region. Failure involved slightly less than half of the free height of the specimens. Significant differences were found in the morphometric parameters calculated in the failure and in the non-failure regions. The cross-sections containing minimum BV/TV values were those most often located inside the failure region (83%, p<0.001). Regression analysis confirmed that variations in BV/TV best describe variations in ultimate stress (R2=0.84) out of the averaged morphometric parameters. The prediction of ultimate stress increased when minimum or maximum values of the morphometric parameters were taken, with the highest prediction found by considering the minimum BV/TV (R2=0.95). In conclusion, due to the heterogeneity of cancellous bone, there may exist regions characterized by a different microarchitecture, where the bone is weaker and consequently is more likely to fail. These regions mostly contain minimum values in BV/TV, which were found to predict ultimate stress better than average BV/TV.

Structural parameters and mechanical strength of cancellous bone in the femoral head in osteoarthritis do not depend on age.

For normal bone, aging has been associated with a decrease of both density and failure strength, and with the development of pathologies such as osteoporosis. Conversely, it has been reported that another common disease, osteoarthritis, may alter these age-related changes in cancellous bone, suggesting that it may have a protective role against osteoporosis and the correspondent fracture risk. It was reported that in the principal compressive region of the femoral head in osteoarthritis the bone density does not depend on age. However, it is not clear if this independence on age of the cancellous bone density corresponds also to a reduced dependence on age of the strength to failure. The present work examined cancellous bone from the principal compressive region of the femoral head of 37 patients having severe osteoarthritis. The aim was (1) to investigate the dependence on age of both the structural parameters and the ultimate stress and (2) to investigate the relationships between the ultimate stress and the structural parameters. Using X-ray microcomputed tomography, three-dimensional structural parameters, such as bone volume fraction, direct trabecular thickness and structure model index were calculated. Then the specimens were compressed to failure to determine the ultimate stress. It was found that none of the investigated structural parameters did depend on age, and also the ultimate stress did not depend on age (p>0.05 for all regressions on age). In addition, the ultimate stress was significantly correlated with the structural parameters, primary with the minimum bone volume fraction and the average bone volume fraction (R(2)=0.95 and R(2)=0.84, respectively). These findings show that severe osteoarthritis or a related factor may change the age dependences of both the structural parameters and the mechanical properties usually reported for normal cancellous bone. These results suggest for this pathology to have a protective role against the age-related decrease in density, the age-related deterioration of the microarchitecture and the age-related decrease of the failure strength for the cancellous bone in the principal compressive region of the human femoral head.

Safety and efficacy of an oxycodone vaccine: Addressing some of the unique considerations posed by opioid abuse.

Among vaccines aimed at treating substance use disorders, those targeting opioids present several unique medication development challenges. 1) Opioid overdose is a common complication of abuse, so it is desirable for an opioid vaccine to block the toxic as well as the addictive effects of opioids. 2) It is important that an opioid vaccine not interfere with the action of opioid antagonists used to reverse opioid overdose or treat addiction. 3) Some opioids are immunosuppressive and chronic ongoing opioid use could interfere with vaccine immunogenicity. 4) Although antibody-bound oxycodone is unable to enter the brain because of its size, it might still be able to activate peripheral opioid receptors. To assess vaccine impact on opioid toxicity, rats vaccinated with oxycodone conjugated to keyhole limpet hemocyanin subunit dimer (OXY-dKLH) adsorbed to alum or controls vaccinated with dKLH were compared with regard to oxycodone-induced hotplate analgesia and oxycodone-induced respiratory depression and bradycardia. Vaccination shifted the dose-response curves to the right, representing protection, for each of these endpoints. Naloxone was equally effective in both OXY-dKLH and control groups, providing complete and rapid reversal of respiratory depression. The administration of a long-acting naltrexone formulation during vaccination did not impair vaccine immunogenicity in mice. Similarly, serum anti-oxycodone antibody titers were not altered by continuous morphine infusion during vaccination compared to opioid-naïve controls. Competitive ELISA assay showed negligible or low affinity of immune antiserum for endogenous opioids or opioid antagonists. In vitro receptor binding assays showed that antibody-bound oxycodone does not activate mu opioid receptors. These data support further study of OXY-dKLH as a potential treatment for oxycodone abuse and suggest that vaccination might also reduce the severity of oxycodone overdose.

Radiopacity of tantalum-loaded acrylic bone cement.

Radiopacifying agents are commonly added to bone cements to enhance the visibility of the cement in radiography. The radiopacifiers usually employed may, however, have undesired effects on the mechanical properties of the cement. A potentially new radiopacifier is tantalum, which in the present work was evaluated in terms of radiopacity. Bone cements containing different percentages of tantalum were compared with plain bone cement as well as with formulations containing different percentages of the commonly used radiopacifier barium sulphate. The radiopacity was assessed quantitatively and qualitatively, by measuring with a digital densitometer the optical density of the cement on X-ray films, and consulting the expertise of ten orthopaedic surgeons. It was found that tantalum does present radiopacity, but not as high as barium sulphate under the specific conditions applied to this study.

Proton magnetic relaxation in bone marrow related to age and bone mineral density: low-resolution in vitro studies.

Detailed analysis of proton spin-spin and spin-lattice relaxation behaviors of the bone marrow in the presence of trabecular bone network was performed at low-resolution (B(0) = 0.496T) on rat vertebrae specimens deprived of spinal cord. Two groups of samples, from young and old healthy animals, were investigated before cellular necrosis had started. BMD measurements were carried out to quantify the expected age-related modifications of the trabecular bone network. 1H-MR measurements were also performed on the same samples, deprived of marrow and saturated with water, in order to control the validity of a possible interpretation of the marrow 1H-MR characteristics, in terms of marrow components, and to investigate the possible employment of these samples to study the trabecular bone network properties. We pointed out that: 1) a bimodal distribution of T(2i) and T(1i) values (distinguishing "fast" and "slow" relaxations) describes satisfactorily all the 1H-MR experimental decays; 2) age-related modifications of the trabecular bone network are marked by correlate variations of the BMD value and of the proton spin-spin relaxation rates in water saturated samples; 3) age-related modifications of marrow are underlined by variations of the average value of the "fast" T(2i) and of the "slow" T(1i) relaxation time distributions, which could be attributed to the marrow components different from the fat granules of the adipose cells. Our results suggest that studies in vitro on bone tissue, by 1H-MR techniques at low-resolution, may contribute to a better bone function characterization and, therefore, to a better clinical utilization of MRI techniques.

Risk of fracture in elderly patients: a new predictive index based on bone mineral density and finite element analysis.

Hip fracture is more and more frequent in elderly population. For this reason, an increasing attention has been focused on the development of a non-invasive method to predict femoral neck fracture. A conventional approach to fracture diagnosis is the measurement of bone mass by dual-energy X-ray absorptiometry in some regions of interest. The aim of this work is to assess a method that accounts for the structural details of the bone providing a more direct determination of strength properties, and improving the diagnostic power of the current densitometric systems. A 2D finite element model of the proximal femur is derived from dual-energy X-ray absorptiometry data. Initially, the method is validated in vitro using a replica of the human femur. The predicted results are compared to strain-gauge measurements and to a 3D finite element model, with good agreement being observed. Then, an in vivo preliminary study on a limited group of patients is carried out. The loading condition that simulates a fall to the side onto the greater trochanter from standing height is employed. All simulations show a peak strain at the femoral neck region with a strain distribution typical of a fall on the side. The proposed method seems to supply a useful tool for the in vivo analysis of the risk of hip fracture.

A computerized system for radiographical evaluation in total hip arthroplasty.

In the field of orthopaedic surgery radiographical image evaluation is frequently used to determine possible pathological processes. For total hip arthroplasty in particular, it is important to evaluate both preoperative femoral morphology and post-operative prosthesis performance in terms of bone adhesion to the implant and, consequently, loosening or stability of prosthesis components. References to a method called Roentgen stereophotogrammetric analysis (RSA) exist in the literature; however, the authors note that this method is expensive and requires the insertion of markers during the operation. Here we present an alternative computerized method for radiographical evaluation which we call radiographical evaluation system in total hip arthroplasty (RESTHA). The accuracy and the repeatability of the method have been evaluated using a medium-sized pelvis-femur system made of composite material. A mean error of +/- 2 mm has been associated to each experimental point. A post-operative inquiry case study is presented to indicate the applicability of the method.

The Telemedicine benchmark--a general tool to measure and compare the performance of video conferencing equipment in the telemedicine area.

In this paper, we describe the 'Telemedicine Benchmark' (TMB), which is a set of standard procedures, protocols and measurements to test reliability and levels of performance of data exchange in a telemedicine session. We have put special emphasis on medical imaging, i.e. digital image transfer, joint viewing and editing and 3D manipulation. With the TMB, we can compare the aptitude of different video conferencing software systems for telemedicine issues and the effect of different network technologies (ISDN, xDSL, ATM, Ethernet). The evaluation criteria used are length of delays and functionality. For the application of the TMB, a data set containing radiological images and medical reports was set up. Considering the Benchmark protocol, this data set has to be exchanged between the partners of the session. The Benchmark covers file transfer, whiteboard usage, application sharing and volume data analysis and compression. The TMB has proven to be a useful tool in several evaluation issues.

Technical validation of low-cost videoconferencing systems applied in orthopaedic teleconsulting services.

Investigation on the applicability of low-cost videoconferencing (VC) for health care services is becoming a real need. Reduced resources drive the administrators to evaluate inexpensive solutions for telemedicine. Considering this scenario, this work is a preliminary step to validate, from a technical point of view, if low-cost VC systems could be suitable for orthopaedic teleconsulting services. For this purpose, four different videoconferencing systems were tested. Each VC system was composed of a computer and a VC device installed in. VC devices were chosen among the most popular and distributed products (made by Intel, PictureTel and Aethra). The Telemedicine Benchmark, a specific tool defined by the authors, was applied to measure the overall systems performances in terms of time delays during basic rate ISDN connections (128 Kbit/s). Results showed that it is possible to apply low-cost videoconferencing systems for orthopaedic teleconsulting services. Most of the systems provided acceptable performance for medical image visualization and real time joint working. Further developments are recommendable to enhance the VC software tools capabilities and to improve software-user interface. reserved.

A study of the application sharing capabilities in telemedicine.

The main aim of this study was to find out if the image format (TIFF or JPEG) influenced the time delay for transferring radiological images by the application sharing tool of a desktop videoconferencing system. The second task of the study was to define a procedure that optimized the time delay to load and remotely visualize the images. The results were achieved by applying a test procedure called 'benchmark protocol'. The videoconferencing system used for the test was Intel ProShare 200 v2.0. The image transfer was performed by a BRI ISDN connection. We showed that the image format had no significant influence on the time delay. We presented an optimal procedure for image transfer. Furthermore, store and forward procedures with simple file transfer were shown to be inferior to the use of application sharing. For radiological image transfer we recommend to use lossless file formats and application sharing with the image already loaded in because this method achieves the lowest time delays.

Topical treatment of experimental skin lesions in rats: macroscopic, microscopic and scanning electron-microscopic evaluation of the healing process.

The effects of the topical antiseptic agents mercurochrome and silver nitrate and of sodium hyaluronate on the process of wound healing were assessed by macroscopic examination, light microscopy histology, electron microscopy and scanning electron microscopy of wounds made by cutting or by abrasion of the skin of rats. It was found that wounds treated with hyaluronic acid underwent longer periods of inflammation (proportional to the presence of mastocytes) and greater amounts of collagen deposition than wounds that were either untreated controls or treated with silver nitrate or mercurochrome, but this did not impede the formation in the early stages of healing of bridges of fibrin between the edges of the wounds.

The effect on the fatigue strength of bone cement of adding sodium fluoride.

New bone cements that include several additives are currently being investigated and tested. One such additive is sodium fluoride (NaF), which promotes bone formation, facilitating implant integration and success. The influence of NaF on the fatigue performance of the cement as used in biomedical applications was tested in this paper. In fact fatigue failure of the cement mantle is a major factor limiting the longevity of a cemented implant. An experimental bone cement with added NaF (12 wt%) was investigated. The fatigue strength of the novel bone cement was evaluated in comparison with the cement without additives; fatigue tests were conducted according to current standards. The load levels were arranged based on a validated, statistically based optimization algorithm. The curve of stress against number of load cycles and the endurance limit were obtained and compared for both formulations. The results showed that the addition of NaF (12 wt%) to polymethylmethacrylate (PMMA) bone cement does not affect the fatigue resistance of the material. Sodium fluoride can safely be added to the bone cement without altering the fatigue performance of the PMMA bone cement.

Comparison of asynchronous and realtime teleconsulting for orthopaedic second opinions.

We studied a teleconsulting service for second opinions in orthopaedics. Three units of the national insurance organization for accidents at work were connected to a large orthopaedic hospital in Bologna. During a 20-month study, 65 consultations were provided: 51 (78%) by asynchronous (store-and-forward) consulting and 14 (22%) by realtime videoconferencing. All the consultations made use of radiology images (radiographs, computerized tomography scans, magnetic resonance imaging scans and ultrasound scans). Video-messages and still images were commonly used to support the asynchronous consultations. More data were transmitted on average for an asynchronous teleconsultation (8 MByte) than in a videoconference (5 MByte). The average time spent by orthopaedic specialists was slightly longer in videoconferences (21 min, SD 8) than in asynchronous teleconsultations (19 min, SD 8). The clinicians confidence in their diagnosis was generally good but was lower in asynchronous consultations. The main problem affecting the telemedicine service was the lack or the low quality of the information received from the referring sites. The clinical complexity of the case and the organizational requirements were declared to be the main factors affecting the choice of consulting procedure. The study showed that the asynchronous method was preferred in the majority of cases and could be easily integrated into clinical practice, although there were some concerns about the diagnostic quality of the information transmitted.

Low-cost ISDN videoconferencing equipment for orthopaedic second opinions.

We validated the application of low-cost ISDN videoconferencing equipment for telemedicine. A telemedicine benchmark was designed and four different office videoconferencing systems were evaluated, all using basic-rate ISDN connections (128 kbit/s). All the low-cost systems showed generally good or acceptable performance for clinical use. We also investigated the feasibility of videoconferencing for an orthopaedic second-opinion service. Eight point-to-point conferences were conducted to discuss real clinical cases by use of interactive sharing of medical images. The average duration of each session was 35 min. Encouraging results were obtained.