Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

INTRODUCTION: BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. AIM: The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. METHODS: Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. RESULTS: Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. CONCLUSION: Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A.

In situ synthesis of calcium phosphate-polycaprolactone nanocomposites with high ceramic volume fractions.

Biodegradable calcium phosphate-PCL nanocomposite powders with unusually high ceramic volume fractions (80-95%) and uniform PCL distribution were synthesized by a non-aqueous chemical reaction in the presence of the dissolved polymer. No visible polymer separation occurred during processing. Depending on the reagents combination, either dicalcium phosphate (DCP) or Ca-deficient HA (CDHA) was obtained. CDHA-PCL composite powders were high pressure consolidated at room temperature yielding dense materials with high compressive strengths. Such densification route provides the possibility of incorporating drug and proteins without damaging their biological activity. The CDHA-PCL composites were tested in osteoblastic and endothelial cell line cultures and were found to support the attachment and proliferation of both cell types.

OFD II, OFD VI, and Joubert syndrome manifestations in 2 sibs.

We present 2 sibs with manifestations of oral-facial-digital syndromes (OFD) and Joubert syndrome. The index patient was the 5th child of healthy nonconsanguineous Turkish parents. At birth this female patient had large hydrocephalus, hypertelorism, deep-set eyes, nystagmus, broad mouth, thick oral frenula, cleft palate, hamartomas of the tongue, postaxial polydactyly of fingers, normal toes, and hypotonia. Cranial MRI showed hydrocephalus and Dandy-Walker malformation. The child had no psychomotor development, was unable to swallow and had severe seizures. She died at 2 months of recurrent apneic episodes. At birth the brother of the index patient showed prominent forehead, broad, deep nasal bridge, cleft palate, multiple hamartomas of the tongue, irregular alveolar ridges, retrognathia, bilateral postaxial polydactyly of the hands and feet, and broad halluces. He had an abnormal breathing pattern with phases of tachypnea and apnea. Cranial MRI showed hydrocephalus, hypoplasia of the cerebellar vermis, Dandy-Walker malformation, and hypomyelination of the corpus callosum. Renal ultrasonography demonstrated multiple small cysts. Ocular fixation was absent and he had a mild nystagmus.

Simpson-Golabi-Behmel syndrome with severe cardiac arrhythmias.

We report on a family with 2 affected males with the X-linked Simpson-Golabi-Behmel (SGB) syndrome. The propositus was a 33-year-old man with pre- and postnatal overgrowth, "coarse" face with hypertelorism, broad nose, wide mouth, malposition of teeth, submucous cleft, accessory nipples, broad hands with hypoplastic index finger nails, and operated left postaxial hexadactyly. From the age of 26 years he suffered from severe tachyarrhythmias, requiring recurrent defibrillations. The brother of the propositus was macrosomic at birth and had a similar facial appearance. In addition he had a pyloric stenosis and a 3/6 systolic murmur. He died at age 4 months. Cardiac defects and conduction disturbances are major components of the SBG syndrome and can be responsible for death in early infancy and perhaps for cardiac arrest in the adult.

Roussy-Lévy syndrome is a phenotypic variant of Charcot-Marie-Tooth syndrome IA associated with a duplication on chromosome 17p11.2.

The Roussy-Lévy syndrome (MIM #180800) was described in 1926 as a disorder presenting with pes cavus and tendon areflexia, distal limb weakness, tremor in the upper limbs, gait ataxia and distal sensory loss. We report a family with affected members in four generations, showing these clinical signs of Roussy-Lévy syndrome and a partial duplication at chromosome 17p11.2. This genetic defect is commonly found in patients with the hypertrophic form of the Charcot-Marie-Tooth syndrome. Our finding provides evidence against the Roussy-Lévy syndrome as a distinct entity but suggests a close relation with the Charcot-Marie-Tooth syndrome. What causes the additional features of gait ataxia and essential tremor needs further clarification.

Drug absorption by the respiratory mucosa: cell culture models and particulate drug carriers.

The inhalation route is of increasing interest for both local and systemic drug delivery, including macromolecular biopharmaceuticals, such as peptides, proteins, and gene therapeutics. In addition to appropriate aerosolization for deposition in relevant areas of the respiratory tract, therapeutic molecules may require an advanced carrier system for safe and efficient delivery to their target. Two approaches to obtain novel carrier systems for pulmonary drug delivery are large porous microparticles with a low aerodynamic diameter and lectin-functionalized liposomes. Epithelial cells of alveolar or bronchial origin, obtained either from patient material or from established cell lines, can be grown on permeable filter supports, resulting in polarized monolayers with functional intercellular junctions. With such in vitro models, transport of drugs into pulmonary epithelial cells and/or across the air-blood barrier, as well as the effect and efficacy of novel drug carrier systems can be systematically studied.

[Survival rate of the cemented Charnley total hip endoprosthesis and modifying parameters]. / Uberlebensraten von zementierten Charnley-Hüfttotalendoprothesen und ihre Beeinflussungsparameter.

AIM OF THE STUDY: To analyse survival of cemented Charnley prostheses, and influencing factors. MATERIAL AND METHODS: 539 patients (mean age: 62 years) undergoing 426 primary, and 200 revision Charnley hip arthroplasties, were examined. The indications for primary operation was idiopathic coxarthrosis in 239 cases (56.1%), and rheumatic coxarthrosis in 56 cases (13.1%). Reasons for revision surgery included aseptic loosening in 123 cases (61.5%), septic loosening in 36 cases (18%), and prosthetic fracture in 13 cases (6.5%). RESULTS: After a mean follow-up of 4.4 years, patients were scored using the Merle d'Aubigne evaluation. The average postoperative score for primary operations was 14.8 points, and for revision surgery 13.1 points. The respective pre-operative scores were 8.2 and 8.1. Radiographic investigation revealed peri-articular calcifications in 28.4% of the primary operations, and in 28.5% of the revisions. Radiolucent lines or protrusions were found in 29.8% of the primary, and in 42.0% of the revisions. At follow-up, 45 implants in 41 patients had been replaced. Kaplan-Meier survival rates for the primary group were 99.6% after one year, 92.8% after five years, 78.3% after 10 years, and 45.9% after 15 years. The respective figures for the revision group were 98.2%, 85.3%, 59.7% and 31.3%. The largest difference was seen in the eleventh year. Patient age had no significant influence for the first 12 years. In the primary group, body mass index showed a significant influence only after 15 years. Analysis of the indications for surgery revealed a slightly higher survival rate in the arthrosis group (84%) vis-a-vis the dysplasia group (83.3%), and a significantly higher rate vis-a-vis patients with rheumatoid arthritis (42%). CLINICAL RELEVANCE: Such factors as age, diagnosis and weight had an influence only over the long term. Significant differences between primary and revision operations were not found.

[Scanning electron microscopy studies of morphologic changes in ultra-high molecular weight polyethylene materials after various processing steps]. / Rasterelektronenmikroskopische Untersuchungen morphologischer Veränderungen am Werkstoff ultrahochmolekulares Polyethylen (UHMWPE) nach verschiedenen Bearbeitungsschritten.

UNLABELLED: Scanning electron microscopic (SEM) studies were carried out on various orthopaedic implants made of ultra-high molecular weight polyethylene (UHMWPE). Both non-sterilized and gamma-sterilized machined (milled) implants were used. Milled implants showed significant surface irregularities, and cross-sections also revealed irregularities in deeper layers of the polyethylene. In gamma-sterilized implants, cracks measuring 10-30 microns in length were found at a depth of 1.5 to 2.5 mm. Crater-like defects were also occasionally seen. CLINICAL RELEVANCE: On the basis of these findings, direct compression moulding and a sterilization process in which oxygen is eliminated can be recommended as ways of improving the quality of UHMWPE used for orthopaedic implants.

[Is Hylamer a suitable substance for use in endoprosthetics?]. / Ist Hylamer ein geeigneter Werkstoff für die Verwendung in der Endoprothetik?

UNLABELLED: In the present study, original UHMWPF implants obtained from different manufacturers were evaluated using Differential Scanning Calorimetry (DSC), Infrared Spectroscopy and Wide Angle X-ray Scattering (WAXS). DSC measurements revealed clear differences in the crystallinity of the implants with figures varying between 58.2% and 73.2%. The highest level of crystallinity (73.2%) was found in the Hylamer material. The results of IR spectroscopy confirmed these findings WAXS investigations showed that Hylamer has both a orthorrhomboid and a monoclinic phase. Remelting was followed by a disappearance of the ortherhomboid phase. CLINICAL RELEVANCE: The use of Hylamer that has been processed by secondary companies is not recommended. The product Hylamer makes clear how the original crystal structure of UHMWPE can be altered. The clearly increased crystallinity in Hylamer also increases the risk of implant failure due to brittleness.

[Effects of different sterilization procedures on degree of oxidation of ultra-high molecular weight polyethylene]. / Auswirkungen unterschiedlicher Sterilisationsverfahren auf den Oxidationsgrad von ultrahochmolekularem Polyethylen (UHMWPE).

We investigated the influence of different sterilization techniques on the degree of oxidation of UHMWPE. Oxidation effects were documented by infrared spectroscopy. The UHMWPE was sterilized with the aid of gamma radiation (2.5 Mrad) in air, gamma radiation in argon, ethylene oxide and autoclaving. Non-sterilized UHMWPE served as control material. The results showed significant differences for the various sterilization techniques employed. The highest degree of oxidation was seen after autoclaving, but gamma radiation was also associated with a significant degree of oxidation. Using gamma radiation in argon, the degree of oxidation was significantly reduced. Following sterilization with ethylene oxide, no oxidation was to be found. In view of these facts, UHMWPE should be sterilized only with ethylene oxide or with gamma radiation in argon.

[Crystalline structure of endoprosthetic UHMWPE in relation to sterilization and mechanical processing]. / Untersuchungen zur Kristallinität des in der Endoprothetik verwendeten UHMWPE in Abhängigkeit von Sterilisierung und Verarbeitung.

We analysed the changes in crystallinity in sterilized and machined UHMWPE products used in the manufacture of total joint components. For this purpose, so-called DC measurements of absolute crystallinity and infrared spectroscopy for determining the relevant crystallinity were carried out. The DSC measurements are based on a further development of differential thermo-analysis (DTA), and a comparison of the melting enthalpy of an unknown material in comparison with known material. The results show particularly high values for gamma sterilized products, and the lowest values, i.e. the least brittleness, after sterilization with ETO. The crystallinity of the original material was 55%, that after gamma sterilized 56.5%, and after steam autoclaving 57.9%; after gamma sterilized in argon 56%, and after ETO sterilized 57.7%. During the period prior to the manufacture of the end product, a wide range of values is determinable. The average crystallinity of the end products varies between 55.4% and 61.6%. Among other things, changes in crystallinity during storage and after implantation must be taken into account. To summarize, we recommended ethylene oxide sterilization, after which the best values of crystallinity were seen in both semi-finished and finished products (prostheses).

[Oxidation of ultra high molecular weight polyethylene as a result of sterilization]. / Oxydation du polyéthylène de très haut poids moléculaire lors de la stérilisation.

PURPOSE OF THE STUDY: To improve (UHMWPE) Ultra High Molecular Weight Polyethylene quality for use in arthroplastic components, sterilization related oxidative changes were investigated in raw and manufactured material. MATERIAL: To evaluate sterilization related effects, 15 x 15 mm samples of UHMWPE were taken from a defined area of raw manufactured UHMWPE plates. The raw manufactured plates were produced using the compression molding method. For sterilization, gamma irradiation with and without air, ETO-sterilization and autoclave sterilization were performed. METHODS: Infrared spectroscopy was used to detect oxidation damage of the implant in superficial and deep layers of the material. RESULTS: Gamma sterilization on air showed 40% more oxidation compared with air-free sterilization. The superficial oxidation on air was 0.33 and showed the lowest value without air on argon with 0.155. Using ethylenoxide sterilization, no great changes in superficial oxidation (0.229) were observed; the smallest change (0.07) was observed at a depth of 2.79 mm. Autoclave sterilization caused the greatest superficial oxidation with 0.622 but low oxidation at 0.094 mm depth. In all samples, further oxidation and ongoing crystallinity was seen with increasing storage time. CONCLUSION: Ethylenoxide sterilization should be recommended for UHMWPE since oxidative changes is lowest. Gamma sterilization can only be recommended without air and at low doses.

Hallermann-Streiff Syndrome: No Evidence for a Link to Laminopathies.

Hallermann-Streiff syndrome (HSS) is a rare inherited disorder characterized by malformations of the cranium and facial bones, congenital cataracts, microphthalmia, skin atrophy, hypotrichosis, proportionate short stature, teeth abnormalities, and a typical facial appearance with prominent forehead, small pointed nose, and micrognathia. The genetic cause of this developmental disorder is presently unknown. Here we describe 8 new patients with a phenotype of HSS. Individuals with HSS present with clinical features overlapping with some progeroid syndromes that belong to the laminopathies, such as Hutchinson-Gilford progeria syndrome (HGPS) and mandibuloacral dysplasia (MAD). HGPS is caused by de novo point mutations in the LMNA gene, coding for the nuclear lamina proteins lamin A and C. MAD with type A and B lipodystrophy are recessive disorders resulting from mutations in LMNA and ZMPSTE24, respectively. ZMPSTE24 in addition to ICMT encode proteins involved in posttranslational processing of lamin A. We hypothesized that HSS is an allelic disorder to HGPS and MAD. As the nuclear shape is often irregular in patients with LMNA mutations, we first analyzed the nuclear morphology in skin fibroblasts of patients with HSS, but could not identify any abnormality. Sequencing of the genes LMNA, ZMPSTE24 and ICMT in the 8 patients with HSS revealed the heterozygous missense mutation c.1930C>T (p.R644C) in LMNA in 1 female. Extreme phenotypic diversity and low penetrance have been associated with the p.R644C mutation. In ZMPSTE24 and ICMT, no pathogenic sequence change was detected in patients with HSS. Together, we found no evidence that HSS is another laminopathy.

Influence of polymer content in Ca-deficient hydroxyapatite-polycaprolactone nanocomposites on the formation of microvessel-like structures.

Calcium phosphate (CaP) ceramics are widely used in bone tissue engineering due to their good osteoconductivity. The mechanical properties of CaP can be modified by the addition of small volume fractions of biodegradable polymers such as polycaprolactone (PCL). Nevertheless, it is also important to evaluate how the polymer content influences cell-material or cell-cell interactions because of potential consequences for bone regeneration and vascularization. In this study we assessed the general biocompatibilty of Ca-deficient hydroxyapatite (CDHA)-PCL disks containing nominally 11 and 24% polycaprolactone using human umbilical vein endothelial cells and human primary osteoblasts. Confocal microscopy showed that both CDHA-PCL variants supported the growth of both cell types. In terms of the endothelial cells grown on CDHA-PCL nanocomposites with 24% PCL, an increased expression of the endothelial marker vWF compared to CDHA-PCL with 11% PCL was observed in real-time polymerase chain reaction analysis. In addition to monocultures, co-cultures of outgrowth endothelial cells, derived from peripheral blood, and primary osteoblasts were assessed as an example of a more complex test system for bone regeneration and vascularization. Constructs based on CDHA with different PCL contents were investigated with regard to the formation of microvessel-like structures induced by the co-culture process using confocal microscopy and quantitative image analysis. Furthermore, the osteogenic differentiation of the co-culture was assessed. As a result, more pre-vascular structures were observed after 1 week on the CDHA-PCL disks with 24% PCL, whereas after 4 weeks of culture the extent of microvessel-like structure formation was slightly higher on the CDHA with 11% PCL. In contrast to this, variation of PCL content had no effect on the osteogenic differentiation in the co-culture.

[Peri-articular calcinosis. Tumor simulating calcinosis and differential diagnosis]. / Periartikuläre Verkalkungen. Die tumoröse Kalzinose und ihre Differentialdiagnose.

The disease of tumorous calcinosis is presented in order to one case and a survey of literature. The epidemiology and etiology are discussed, because of a rare manifestation of metabolic disorders. The differential diagnosis includes genetic disorders, recurrent soft-tissue trauma and renal failure. The clinical symptoms like swelling or deficit of function as well as radiologic appearances may lead to the diagnosis. The way of treatment is given by radical excision and metabolic supervision. The multiloculated presentation of periarticular, dental, subcutaneous and other lesions is pointed out.

Is Hylamer adequate for use in alloarthroplasties?

In the present study ultrahigh molecular weight polyethylene (UHMWPE) implants from different manufactures were evaluated by differential scanning calorimetry (DSC), infrared spectroscopy (IRS) and wide-angle X-ray scattering (WAXS). With DSC measurements the crystallinity of all evaluated implants ranged between 58.2% and 73.2%. The highest crystallinity was documented in Hylamer with 73.2%. These results were checked and confirmed by infrared spectroscopy. According to WAXS studies Hylamer had an orthorhomboid phase as well as a monocline phase. After remelting the orthorhomboid phase was no longer detectable. We do not recommend using UHMWPE implants which have been secondarily modified. The product Hylamer clearly demonstrates how the original crystal structure of UHMWPE can be altered. The described changes in Hylamer implants will probably lead to an increased failure rate.

[Results of long-term therapy of chronic, post-traumatic osteomyelitis with gentamycin PMMA chains]. / Langfristige Therapieergebnisse der chronischen, posttraumatischen Osteomyelitis mit Gentamycin-PMMA-Ketten.

From 1977 to 1983, 173 patients suffering from chronic posttraumatic osteomyelitis were treated by local debridement and implantation of septopal beads; 102 patients were subsequently evaluated after follow-up periods of between 4 and 10 years. In 89.2% of these cases cure was achieved during the initial hospital stay. In 15.7% further debridements were necessary during the initial hospital stay to manage infection. In 91 patients there was no early recurrence until 1983. After 1983 only 7 patients had late recurrences. At the time of follow up 95 patients (94.5%) did not show any signs of infection. It was necessary for 9 patients to change their profession because of the disease. Full use of the extremity involved was reported by 45 patients, and some slight limitations by 46. In 11 cases the patients could not use the extremity involved in activities of daily living. In mono-infections (87.2%) and in infection with multiple different pathogen chains (82%) Staphylococcus aureus was by far the most common pathogen. Our findings indicate that both the outcome and the prognosis score are significantly better after the treatment regimen used than after other treatment modalities.

Debris removal by head-pushing in A. florea Fabr. honeybees.

The nest of the dwarf honeybee A. florea Fabr. consists of a single comb attached to a tree branch. Recruitment dances take place on the upper surface of the comb that must therefore be kept clear of debris. We report here, for the first time, a behaviour that serves for removing leaves and other foreign objects from the surface of the comb. Individual workers crawl under the object and lift it with their heads, pushing it towards the rim where it eventually slides off the comb. Objects that are heavier or fixed at one end such as leaves are nevertheless lifted and kept away from the surface for up to several minutes. This "head-pushing" is frequently performed without the aid of mandibles, and individuals performing it maintain a distinctive posture, holding the forelegs at an angle without touching the object. Repeated involvement of particular individuals indicate that head-pushers might form a distinct task group.

[Changes in crystallinity by sterilization and processing of ultrahigh molecular polyethylene used in endoprosthetics]. / Kristallinitätsveränderungen durch Sterilisierung und Verarbeitung des in der Endoprothetik verwendeten ultrahochmolekularen Polyethylens.

PURPOSE OF THE STUDY: We wanted to analyze crystallinity changes during sterilization in raw and in UHMWPE components for total joint arthroplasty manufactured. MATERIALS AND METHODS: To analyze the crystallinity DSC measurements and to determine the relative crystallinity infrared spectroscopy was performed. The DSC measurements were based on a comparison between the melting enthalpy of one known material to the unknown material. RESULTS: The raw manufactured non-sterilized sample showed a value for the DSC-measured crystallinity of 55.5%; the sample after gamma-sterilization without air was 56.0%, and with air 56.5%. The crystallinity after ethylene-oxide sterilization was 57.7% and after autoclavation 57.9%. The manufacturing time from the raw to the arthroplasty part showed some what different results. The average crystallinity of the samples differed between 55.4% and 61.6%. The best results were achieved in ETO-sterilized samples and one gamma-sterilized sample without air. The highest values were seen for the Hylamer. CONCLUSION: In summary, the best values for crystallinity were seen in both raw and completed arthroplasty products with ETO sterilization. For medical use cancerogenous factors must be excluded. Gamma-sterilization without air can be accepted as well. Increasing the crystallinity too much can lead to more wear debris.

[Molecular characterization of tissue-engineered articular chondrocyte transplants based on resorbable polymer fleece]. / Molekulare Charakterisierung von gezüchteten humanen dreidimensionalen Chondrozytentransplantaten.

Three-dimensional arrangement and subsequent transplantation of chondrocytic cells in resorbable polymers has been shown to be a promising technique for the treatment of cartilaginous defects. Engineering of artificial cartilage tissue includes dedifferentiation of chondrocytes in monolayer culture, the use of biodegradable matrices and polymer scaffolds, and re-expression of chondrocytic marker genes in three-dimensional culture. The aim of this study was to characterize molecularly the phenotypic changes occurring with autologous cartilage tissue engineering. Human articular chondrocytes were isolated, cultured in medium containing human serum, and expanded up to passage 3. Chondrocytes were embedded in human fibrinogen and in polyglactin-polydioxanon fleeces and cultured three-dimensionally up to 4 weeks. Dedifferentiation of chondrocytes in monolayers and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice was assessed by gene expression analysis of typical chondrocytic genes, histology, and immunohistochemistry. The expansion of chondrocytes with human serum resulted in the induction of type I and type III collagens, whereas cartilage-specific type II collagen, cartilage oligomeric matrix protein, cartilage link protein, and aggrecan were repressed and induced again after three-dimensional arrangement of chondrocytes in polyglactin-polydioxanon. Transplantation experiments documented the synthesis of proteoglycan and cartilage-specific type II collagen in vivo. Three-dimensional arrangement of human articular chondrocytes in resorbable polyglactin-polydioxanon fleeces supports chondrogenic differentiation and the formation of a hyaline-like cartilaginous matrix in vitro and in vivo.