ACL reconstruction is the preferred cost-effective treatment strategy for ACL tears and yields reduced societal costs relative to rehabilitation once indirect cost factors, such as work status and earnings, are considered. The cost of an ACL tear over the lifetime of a patient is substantial, and resources should be directed to developing innovations for injury prevention and for altering the natural history of an ACL injury.

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Amphiphilic gadolinium complexes were investigated as potential magnetic resonance imaging (MRI) contrast agents. A series of complexes was synthesized in order to study the effect of hydrophilic phosphodiester groups on albumin binding, relaxivity, and blood half-life in rats. Thus, compound 11a, a diethylenetriaminepentaacetato aquo gadolinium(III) (Gd-DTPA) derivative with an octyl substituent, was synthesized and compared to 5b, the analogous octyl derivative containing a phosphodiester linkage between the gadolinium chelate and the alkyl chain. Likewise, 11b, a naphthyl Gd-DTPA derivative, was compared to the naphthyl phosphodiester derivative 5c. A direct comparison is not available for 5a, a 4,4-diphenylcyclohexyl phosphodiester Gd-DTPA derivative; however, its pharmacokinetic properties mirror those of the other phosphodiester derivatives. Although the introduction of the phosphodiester moiety decreased log P by approximately 1.7 units, albumin binding data obtained in 4.5% human serum albumin (HSA) indicated that derivatives containing the phosphodiester group exhibited somewhat higher albumin affinity than their alkyl analogues (54 +/- 5 and 44 +/- 4% for 5b and 11a, respectively; 40 +/- 4 and 30 +/- 3% for 5c and 11b, respectively). Both classes of agents were characterized by enhanced relaxivity in the presence of 4.5% HSA (r1 = 16-42 mM(-1) s(-1) at 20 MHz and 37 degrees C) as compared with the relaxivity values measured in phosphate-buffered saline (PBS) alone (r1 = 4.6-6.6 mM(-1) s(-1) at 20 MHz and 37 degrees C). Pharmacokinetic data indicated that compound 5b had a half-life of 14.3 +/- 1.8 min in the rat as compared with a half-life of 6.20 +/- 0.04 min for the non-phosphodiester analogue 11a. Similarly, the half-life obtained for the phosphodiester 5c was 14.3 +/- 1.7 min as compared with a half-life of 6.80 +/- 0.03 min for 11b. The percent biliary excretion was significantly lower for the phosphodiester compounds than for non-phosphodiester analogues (17.7 +/- 4.0 and 66.9 +/- 3.4% for 5b and 11a, respectively; 17.0 +/- 1.6 and 64.3 +/- 9.0% for 5c and 11b, respectively). The percent biliary excretion (15.8 +/- 4.4%) and plasma half-life in the rat (23.1 +/- 2.9 min) for 5a are consistent with the extended plasma half-life of the other phosphodiester derivatives. Taken together, the enhanced relaxivity and extended blood half-life of the phosphodiester derivatives support the concept of using endogenous albumin binding to achieve blood pool-like properties for small-molecule magnetic resonance imaging (MRI) contrast agents.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule.

Integrin ␣ 4 ␤ 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 ␣ 4 ␤ 1 inhibitors, the anti-rat ␣ 4 monoclonal antibody TA-2 and the small molecule inhibitor. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (K D of Ͻ10 pM) inhibitor of ␣ 4 ␤ 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 ␣ 4 integrin expression on the cell surface, which resulted from internalization of ␣ 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface ␣ 4 ␤ 1 . Our results with BIO5192 indicate that ␣ 4 ␤ 7 does not play a role in this model and that blockade of ␣ 4 ␤ 1 /ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to ␣ 4 ␤ 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of ␣ 4 ␤ 1 integrin, will be a valuable reagent for assessing ␣ 4 ␤ 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.Integrins are a large family of cell surface receptors that mediate cell/cell and cell/matrix interactions and signal transduction. They exist as noncovalent ␣␤ heterodimers of different combinations of ␣ and ␤ chains and share extensive structural homology. The leukocyte integrin ␣ 4 ␤ 1 regulates normal lymphocyte trafficking (Lobb and Hemler, 1994) and provides a key costimulatory signal supporting cell activation (Clark and Brugge, 1995). During inflammatory responses, it regulates lymphocyte migration into the damaged tissues and thus has been recognized as an attractive therapeutic target. In vivo studies using blocking monoclonal antibodies (Lobb and Hemler, 1994) and inhibitory peptides (Molossi et al., 1995) have verified the critical role of ␣ 4 ␤ 1 integrins in leukocyte-mediated inflammation. Numerous EAE models of multiple sclerosis have been designed to recapitulate important aspects of the disease and are responsive to ␣ 4 inhibitors (Yednock et al., 1992). Recent positive phase II data using the anti-␣ 4 antibody 1 Current address: Wyeth, Cambridge, MA.

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