Acute Intravenous Injection Toxicity Study of Escherichia coli-Derived Recombinant Human Bone Morphogenetic Protein-2 in Rat.

STUDY DESIGN: Prospective in vivo toxicity study. PURPOSE: To evaluate the conducted acute toxicity study of Escherichia coli (E. coli)-derived recombinant human bone morphogenetic protein-2 (rhBMP-2) with 6-weeks old Sprague-Dawley rats. OVERVIEW OF LITERATURE: rhBMP-2 has well-known osteoinductivity and it is used as a bone graft substitute. E. coli-derived rhBMP-2 can be mass-produced with relatively low costs. E. coli-derived rhBMP-2 facilitates osteoblastic differentiation and bone formation in vitro and in vivo. However, studies regarding side effects or toxicity of E. coli-derived rhBMP-2 have not been published. Thus, we conducted the acute toxicity study of E. coli-derived rhBMP-2 on 6-weeks old Sprague-Dawley rats. METHODS: One mg of BMP-2 was diluted in 0.285 mL of glycine buffer to prepare high BMP-2 concentrations (3.5 mg/mL). Intermediate (0.9 mg/mL) or low (0.35 mg/mL) concentrations of BMP-2 solution was prepared by serial dilutions. The compound was administrated at a dose of 0, 0.7, 1.8, 7 mg/kg by single intravenous injection to five of male and female rats. After the injection, the gross general observations including changes of body weight and histopathological analysis was performed for 14 days. RESULTS: No animal was found dead during the experiment and the body weight changes were both statistically insignificant in the control and experimental groups. No abnormal sign was shown in general observations and autopsy examinations. CONCLUSIONS: Thus, the lethal dose of E. coli-derived rhBMP-2 should be higher than 7 mg/kg with a single intravenous injection.

The short-term effects of repetitive E. coli-derived rhBMP-2 administration through intravenous injection in rats.

Escherichia coli-derived recombinant human (rh)BMP-2 (E.BMP-2) can be used as a bone graft substitute because to its high osteoinductivity, but its toxicity is not well understood. Thus, we report on the toxicity of E.BMP-2 in Sprague-Dawley rats under the condition of repetitive injection for 2 weeks. Randomly selected 10 male and female rats were administered with E.BMP-2 at a dose of 0.05, 0.18 or 0.5 mg/kg as an experimental group. A control group with another 10 rats was given E.BMP-2 carrier. Both E.BMP-2 and E.BMP-2 carrier were administered through intravenous injection for 2 weeks. For toxicokinetics study, 3 male and female rats were randomly selected from each group. During the observation period, general symptom, weight and food intakes were monitored, and ophthalmic and urine tests were performed as well. After the observation period, all animals were subjected to blood test, biochemical analysis and organ-weight measurement. During autopsy, visual inspections and histopathological examinations were done. Toxicokinetics study confirmed systemic exposure of the test material. No death or abnormal clinical sign was found during the injection period. Toxicity changes induced by the injection were not detected in autopsy or the tests for weight, food intakes, ophthalmology, hematology and serum biochemistry. The female groups administered with 0.18 and 0.5 mg/kg (the female 0.18-mg/kg group and the female 0.5-mg/kg group) showed absolute and relative weight loss in ovaries and reduced corpora lutea. It was the expected pharmacologic activity, rather than toxicity. The histopathological test revealed cartilage formation and increased fibroblast around the tail vein, but these were thought to be the result of osteoinductivity of the test material. In the male group with 0.5 mg/kg of E.BMP-2 (the male 0.5-mg/kg group), local appearance of multinucleated cells in lung parenchyma was observed, but it was considered as the natural reaction to remove E.BMP-2, which is a recombinant protein. In toxicokinetics study, systemic exposure (area under the serum concentration-time curve and maximum observed serum concentration) increased as the injection dose was increased in both male and female rats, and no clear difference was noticed between the sexes. Blood drug content did not change during the injection period, but the half-life was shortened as the injection dose was increased. Under the condition of this study, the no observed adverse effects level of E.BMP-2 was over 0.5 mg/kg in both male and female rats.

Comparative study of Chinese hamster ovary cell versus Escherichia coli-derived bone morphogenetic protein-2 using the critical-size supraalveolar peri-implant defect model.

BACKGROUND: Chinese hamster ovary (CHO) cell-derived recombinant human bone morphogenetic protein-2 (rhBMP-2) has been introduced for spine, long bone, and craniofacial indications. Escherichia coli- (E. coli) derived rhBMP-2 displays comparable efficacy to CHO cell-derived rhBMP-2 in vitro and in small-animal models. The objective of this study is to evaluate the efficacy of E. coli-derived rhBMP-2 compared to the benchmark CHO cell-derived rhBMP-2 using an established large-animal model. METHODS: Contralateral, critical-size supraalveolar peri-implant defects in six adult male Hound Labrador mongrel dogs received CHO cell- or E. coli-derived rhBMP-2 (0.2 mg/mL) in an absorbable collagen sponge (ACS) carrier. In each quadrant, three dental implants were placed. A titanium mesh device was used to support space provision. The animals received fluorescent bone markers for qualitative evaluations. Animals were euthanized at 8 weeks for histopathologic and histometric evaluation. RESULTS: Clinical healing included significant swelling, but none of the animals experienced wound dehiscences. CHO cell- and E. coli-derived rhBMP-2 supported comparable bone formation (new bone area, 35.8 ± 3.6 versus 30.1 ± 2.2 mm(2); bone density, 31.8% ± 1.6% versus 35.6% ± 2.5%; and osseointegration, 32.9% ± 7.4% versus 33.7% ± 8.1%) without statistically significant differences between treatments. Newly formed immature delicate trabecular bone in fibrovascular marrow filled the space underneath the titanium mesh and extended coronally above the mesh. Seroma formation was frequently observed. There were no discernable qualitative histologic differences between treatments. CONCLUSION: CHO cell- and E. coli-derived rhBMP-2 in an ACS carrier appear equally effective at inducing local bone formation in support of dental implant osseointegration.

Promising efficacy of Escherichia coli recombinant human bone morphogenetic protein-2 in collagen sponge for ectopic and orthotopic bone formation and comparison with mammalian cell recombinant human bone morphogenetic protein-2.

Nonglycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in Escherichia coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited owing to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via microcomputed tomography-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells. Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of human mesenchymal stromal cells at similar levels. Similar tendencies were observed in microcomputed tomography analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density, and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 µg) not only induced complete regeneration of an 8-mm critical-sized defect at 4 weeks, but also led to new bone with the same bone mineral density as normal bone at 8 weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 µg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in absorbable collagen sponge may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering.

Stabilizing peptide fusion for solving the stability and solubility problems of therapeutic proteins.

PURPOSE: Protein aggregation is a major stability problem of therapeutic proteins. We investigated whether a novel stabilizing peptide [acidic tail of synuclein (ATS) peptide] could be generally used to make a more stable and soluble form of therapeutic proteins, particularly those having solubility or aggregation problems. METHODS: We produced ATS fusion proteins by fusing the stabilizing peptide to three representative therapeutic proteins, and then compared the stress-induced aggregation profiles, thermostability, and solubility of them. We also compared the in vivo stability of these ATS fusion proteins by studying their pharmacokinetics in rats. RESULTS: The human growth hormone-ATS (hGH-ATS) and granulocyte colony-stimulating factor-ATS (G-CSF-ATS) fusion proteins were fully functional as determined by cell proliferation assay, and the ATS fusion proteins seemed to be very resistant to agitation, freeze/thaw, and heat stresses. The introduction of the ATS peptide significantly increased the storage and thermal stabilities of hGH and G-CSF. The human leptin-ATS fusion protein also seemed to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. Furthermore, the ATS peptide greatly increased the solubility of the fusion proteins. Finally, pharmacokinetic studies in rats revealed that the ATS fusion proteins are also more stable in vivo. CONCLUSION: Our data demonstrate that a more stable and soluble form of therapeutic proteins can be produced by fusing the ATS peptide.

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

Oxidized glutathione stimulated the amyloid formation of alpha-synuclein.

alpha-Synuclein is the major filamentous constituent of Lewy bodies found in Parkinson's disease (PD). The amyloid formation of alpha-synuclein was significantly facilitated by oxidized glutathione (GSSG) as the lag period of the aggregation kinetics was shortened by 2.5-fold from its absence. Reduced glutathione (GSH), on the other hand, did not influence the lag phase although it increased the final amyloid formation. The GSSG stimulation was specific for not only alpha-synuclein but also its intactness. The preferred GSSG interaction of alpha-synuclein to GSH was also demonstrated with dissociation constants of 0.53 and 43.5 mM, respectively. It is suggested that the oxidative stress favoring the GSSG generation from GSH could result in the augmented amyloid formation of alpha-synuclein, which ought to be related to the pathogenesis of PD.

Lipid interaction of alpha-synuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2.

Alpha-synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between alpha-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of alpha-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing beta-synuclein and a C-terminally truncated alpha-synuclein (alpha-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of alpha-synuclein. In the presence of Cu2+ and H2O2, phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of alpha-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H2O2 during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and alpha-synuclein increased from Kd of 44.7 microm to 5.9 microm in the presence of the lipid. Taken together, PI not only affects alpha-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H2O2 are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration.

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes.

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.