Design, synthesis, and analysis of a polyethelene glycol-modified (PEGylated) small molecule inhibitor of integrin {alpha}4{beta}1 with improved pharmaceutical properties.

Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.

An assessment of the mechanistic differences between two integrin alpha 4 beta 1 inhibitors, the monoclonal antibody TA-2 and the small molecule BIO5192, in rat experimental autoimmune encephalomyelitis.

Integrin alpha 4 beta 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of alpha 4 beta 1 inhibitors, the anti-rat alpha 4 monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-[[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino]-4-[4-methyl-2(S)-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (KD of <10 pM) inhibitor of alpha 4 beta 1. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in alpha 4 integrin expression on the cell surface, which resulted from internalization of alpha 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface alpha 4 beta 1. Our results with BIO5192 indicate that alpha 4 beta 7 does not play a role in this model and that blockade of alpha 4 beta 1/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to alpha 4 beta 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of alpha 4 beta 1 integrin, will be a valuable reagent for assessing alpha 4 beta 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.

Malignant melanocytic neural crest tumor in a young chicken (Gallus domesticus).

A malignant melanocytic tumor was found in an 8-week-old chicken. The tumor, which was composed of melanocytes, ganglion cells, nerves, and primitive pressure receptors, was examined by light microscopy, transmission electron microscopy, and immunohistochemistry. Antibodies to neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 protein variably stained nerves, but melanocytes were only rarely immunolabelled by NSE antibodies and there was no specific staining of these cells for S-100 or GFAP. Ultrastructurally, neoplastic melanocytes contained melanin within melanosomes and premelanosomes and did not resemble Schwann cells.

Spontaneous equine pulmonary granular cell tumors: morphologic, histochemical, and immunohistochemical characterization.

Spontaneous equine pulmonary granular cell tumors were diagnosed in six mature horses at slaughter. These tumors were grossly recognized as multiple (5/6) or single (1/6) creamy white, firm nodules. The tumors, located adjacent to bronchi and bronchioles, often invaded airways, resulting in partial to complete occlusion of the lumina. Neoplastic cells were rounded to polyhedral with numerous eosinophilic cytoplasmic granules that reacted uniformly positive with S-100 and neuron-specific enolase antibodies and multifocally with glial fibrillary acidic protein antibodies. These cells were negative for muscle-specific actin, lysozyme, cytokeratin, chromogranin A, and myelin basic protein antigens and did not stain with silver by the Grimelius technique. Uniformly blue-green and scattered pink intracytoplasmic granules were evident with luxol fast blue and periodic acid-Schiff counterstain for myelin and myelin breakdown products. Histochemical and immunohistochemical staining results of these tumors suggest that they are composed primarily of myelinating Schwann cells with lesser numbers of scattered nonmyelinating Schwann cells. The morphologic features of the equine pulmonary granular cell tumors are strikingly similar to those of endobronchial granular cell tumors of human beings.

The effect of ibuprofen on microvascular thrombosis in an experimental rabbit model.

The efficacy of ibuprofen in reducing microvascular thrombosis in a well-established experimental model was studied. Bilateral 2-mm arterial inversion grafts were constructed in the femoral arteries of New Zealand White rabbits. The experimental group (n = 40 grafts) received subcutaneous injections of ibuprofen 15 mg/kg t.i.d. beginning 1 day preoperatively and continued for 7 days postoperatively. The control group (n = 42 grafts) received injections of an equivalent volume of saline three times per day. Patency was evaluated at 7 days by the distal milking test. Seventy-three percent of the ibuprofen grafts were patent at 7 days, whereas 57% of the control grafts remained open. This difference in microvascular patency was not statistically significant. Representative scanning electron micrographs revealed a moderate reduction in aggregated platelets and overall clot density in the patent ibuprofen arterial inversion grafts compared with the patent control specimens. Although the use of ibuprofen as a sole antithrombotic agent cannot be recommended as the result of this study, it may be efficacious when used in conjunction with other agents such as dextran 40.

Contraction and growth of wounds covered by meshed and non-meshed split thickness skin grafts.

Full thickness wounds, one on either side of the backs of rabbits, were covered with meshed or non-meshed split thickness skin grafts. The wounds were measured over a 3 month period and the minimum and maximum sizes of the wounds determined as well as the rate of growth. Wounds covered with meshed grafts contracted to 55% of the original size while those covered with non-meshed grafts contracted to 81.5% (p = less than 0.0001). The meshed wounds grew to 114.6% of original area and non-meshed grew to 153.3% (p = 0.035) after 13 weeks.

The effect of the "patency test" on arterial endothelial surface.

The effect of the "milking patency test" on the arterial endothelium of the experimental rat is investigated. Each of 18 rats served as its own control. Bilateral femoral artery anastomoses were performed in identical fashion. The milking patency test was performed on one side (experimental) but not on the other (control). After removal of the approximator clamps, rats were perfusion-fixed in situ. Harvested experimental vessels stained with silver showed a twofold increase in endothelial cell loss compared to controls. Visual evidence of extensive endothelial loss was seen on silver-stained segments in the tested area. This was corroborated by scanning electron microscopy. The degree of damage seen in this study suggests that the milking patency test is too traumatic for use in clinical microsurgery.

The anatomy of the epigastric flap in the experimental rat.

In order to explain the variability noted in the fluorescein staining characteristics of the epigastric axial flap in the experimental laboratory rat, the anatomy of the blood supply of this particular flap was investigated. In 10 rats, the vascular pattern of the superficial epigastric trunk to the abdominal skin was found to be similar. A small branch of the trunk extended toward the medial abdominal skin to collateralize with a branch of the internal mammary vessel entering from the chest skin. A large extension of the superficial epigastric trunk extended laterally toward the midlateral point of the animal and cephalad to collateralize with the vessels in the lateral chest wall, presumably the lateral thoracic artery. Fluorescein staining characteristics of 10 flaps including or excluding the lateral branch were examined, and a significant difference was found in the immediate fluorescein staining characteristics. The flaps that included the lateral branch fluoresced to an average of 89.2 percent of the flap length, and those in which the lateral branch was excluded fluoresced to 68.7 percent of the flap length. The use of this flap model in experimental flap research should include a precise description of the anatomy of the particular flap used in order to ensure reproducible results.

Prostacyclin production by vein grafts in the arterial circulation: a study in rats.

The in vitro production of prostacyclin by vein grafts was measured using vascular rings followed by radioimmunoassay of the medium. The grafts were prepared by interposing a segment of epigastric vein in a gap in the ipsilateral femoral artery of the rat. Measurements made over a period of 1 to 42 days showed a progressive rise from venous levels (less than 0.2 ng/ml) to values close to the arterial controls (10.9 +/- 2.2 ng/ml). This deficiency of prostacyclin synthesis during the "arterialization" of the graft may account for some of the early failures which can occur in vein grafting and suggests the possibility of pharmacological intervention to improve the success rate.

Vascular labelling with monastral blue B.

Vascular labelling is an established technique of experimental pathology whereby leaky vessels can be identified in vivo. A suspension of a suitable colloidal pigment is injected intravenously; the pigment is then trapped in the wall of the leaky vessels. The colloidal preparation of carbon black, which has been used for many years for this purpose, is no longer commercially available. This communication introduces a substitute: Monastral blue B which gives beautiful preparations in whole mounts, is readily visible in paraffin and plastic embedded histologic sections, has a distinctive appearance in electron micrographs, and is nontoxic in the required dosage.

Brain cell surface antigens detected by anti-corpus callosum antiserum.

When rabbits are injected with tissue homogenates of white matter from bovine corpus callosum, an antiserum is produced which reacts with the surface membrane of 20-30% of all cells obtained by trypsin-dissociation of cerebellum from 10-day-old mice. The antigen or set of antigens recognized by this antiserum is detectable on embryonic, early postnatal, and adult mouse brain, but not on liver, spleen, kidney, thymus and sperm. The antigen is expressed in different regions of the brain and also, in decreased amounts, on retina. In histological sections of cerebellum from 21-day-old mice the antigen is predominantly localized in white matter tracts. Whereas nervous tissue from chicken and rabbit does not carry detectable levels of the antigen, rat, bovine and human brains are antigen-positive.