rhBMP-2/calcium phosphate matrix accelerates osteotomy-site healing in a nonhuman primate model at multiple treatment times and concentrations.

BACKGROUND: While recombinant human bone morphogenetic protein-2 (rhBMP-2) administered in a calcium phosphate cement accelerates osteotomy-site healing in animal models when administered three hours after surgery, definitive fracture treatment is often delayed. The present study evaluated the ability of rhBMP-2, administered in a new particulating calcium phosphate matrix, to accelerate nonhuman primate fibular osteotomy-site healing following treatment at multiple treatment times and concentrations. METHODS: The ability of 1.5-mg/mL rhBMP-2/calcium phosphate matrix to accelerate osteotomy-site healing when administered three hours, one day, one week, or two weeks after surgery was first evaluated with use of bilateral proximal and distal fibular osteotomy sites in adult male monkeys. In a second study, the healing of osteotomy sites that had been treated with the administration of calcium phosphate matrix alone and with different concentrations of rhBMP-2/calcium phosphate matrix (0.5 mg/mL, 1.5 mg/mL, or 4.5 mg/mL) seven days after surgery was compared with that of contralateral, untreated osteotomy sites. In a third study, the histologic progression of osteotomy-site healing following treatment with 1.5-mg/mL rhBMP-2/calcium phosphate matrix or calcium phosphate matrix alone, administered three hours or one week after surgery to the osteotomy site, was assessed at multiple time points for as long as twenty-four months after surgery. RESULTS: Radiographs demonstrated increased callus area and more rapid healing in response to 1.5-mg/mL rhBMP-2/calcium phosphate matrix administered over the range of treatment times after surgery as compared with the findings of previous reports on untreated osteotomy sites. Bone formation appeared at the osteotomy sites sooner following treatment at one and two weeks as compared with the findings at the earlier time-points. Scintigraphic imaging at one day and one week after surgery showed prolonged retention of rhBMP-2 at the osteotomy site following an initial burst release. In the second study, radiographic, peripheral quantitative computed tomographic, biomechanical, and microscopic evaluation demonstrated that administration of 1.5 and 4.5-mg/mL rhBMP-2/calcium phosphate matrix one week after surgery accelerated osteotomy-site healing by 40% to 50% compared with the findings in untreated controls. The magnitude of acceleration was less in response to 0.5-mg/mL rhBMP-2/calcium phosphate matrix, and calcium phosphate matrix alone did not accelerate osteotomy-site healing. Histological evaluation indicated that an increased cellular infiltrate and increased direct bone formation contributed to the accelerated osteotomy-site healing following administration of rhBMP-2/calcium phosphate matrix at one week compared with three hours after surgery. CONCLUSIONS: A single percutaneous injection of rhBMP-2/calcium phosphate matrix accelerated healing in nonhuman primate fibular osteotomy sites over a wide range of treatment times. Efficacy was optimized in association with the administration of 1.5-mg/mL rhBMP-2/calcium phosphate matrix. Delaying treatment for one week further accelerated healing because of an increase in the number of responding cells and an increase in direct bone formation.

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance.

PURPOSE: 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs. METHODS: Doses of 0, 2.4, 12 and 24 mg/m2 per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m2 per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m2 per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5. RESULTS: Daily i.v. administration of 17-DMAG at doses of 24 mg/m2 per day in rats or 16 mg/m2 per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration. CONCLUSIONS: With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m2 per day in rats and 8 mg/m2 per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m2 per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Mechanical environment alters tissue formation patterns during fracture repair.

Fracture repair has previously been shown to be sensitive to mechanical environment, yet the specific relationship between strain character, magnitude and frequency, as well as other mechanical parameters, and tissue formation is not well understood. This study aimed to correlate strain distribution within the healing fracture gap with patterns of tissue formation using a rat model of a healing osteotomy subject to mechanical stimulation in bending. Finite element models based on realistic tissue distributions were used to estimate both the magnitude and spatial distribution of strains within the fracture gap. The spatial distribution of regenerating tissue was determined by microcomputed tomography and histology, and was confirmed using reverse transcription-polymerase chain reaction (RT-PCR). Results suggest that tensile strains suppress chondrogenesis during the mechanical stimulation period. After stimulation ends, however, tensile strains increased chondrogenesis followed by rapid bone formation. In contrast, in compressive environments, bone is formed primarily via intramembranous ossification. Taken together, these results suggest that intermittent tensile strains during fracture repair stimulate endochondral ossification and promote eventual bone healing compared to intermittent compressive strains and unstimulated fractures. Further understanding of these relationships may allow proposal of optimal therapeutic strategies for improvement of the fracture repair process.

The effects of tetrathiomolybdate (TTM, NSC-714598) and copper supplementation on fertility and early embryonic development in rats.

Based on its ability to chelate copper, TTM is being studied as an antiangiogenic agent for cancer therapy. The purpose of this study was to evaluate the toxicity of TTM and the protection of copper supplementation on the reproductive capability of male and female CD rats. Doses of 0, 1, 4, and 12 mg/kg/day with copper supplementation (110 mg/kg of diet) were given by gavage. There were no effects on the estrous cycle or reproductive indices, or maternal toxicity in any female dose group. Male rats given 12 mg/kg/day showed significant decreases in body weight gains and food consumption, and anemia. Serum ceruloplasmin levels were dose-dependently decreased in all male dose groups. Reduced epididymal weights, sperm counts, and sperm motility, sperm morphologic abnormalities and histopathologic changes in testis and epididymis occurred only at 12 mg/kg/day. Dietary copper supplementation prevented the adverse sperm effects produced by 12 mg/kg/day of TTM.

Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry.

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.