Irving Sucholeiki

A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models

OBJECTIVE Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity. METHODS Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors. RESULTS A number of non-hydroxamic acid-containing compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompetitive inhibition, and Dixon plot analysis from competition studies with a zinc chelator (acetoxyhydroxamic acid) and ALS 1-0635 demonstrated nonexclusive binding. ALS 1-0635 inhibited bovine articular cartilage degradation in a dose-dependent manner (48.7% and 87.1% at 500 nM and 5,000 nM, respectively) and was effective in inhibiting interleukin-1alpha- and oncostatin M-induced C1,C2 release in human OA cartilage cultures. ALS 1-0635 modulated cartilage damage in the rat MIA model (mean +/- SEM damage score 1.3 +/- 0.3, versus 2.2 +/- 0.4 in vehicle-treated animals). Most significantly, when treated twice daily with oral ALS 1-0635, rats with surgically induced medial meniscus tear exhibited histologic evidence of chondroprotection and reduced cartilage degeneration, without observable musculoskeletal toxicity. CONCLUSION The compounds investigated in this study represent a novel class of MMP-13 inhibitors. They are mechanistically distinct from previously reported broad-spectrum MMP inhibitors and do not exhibit the problems previously associated with these inhibitors, including selectivity, poor pharmacokinetics, and MSS liability. MMP-13 inhibitors exert chondroprotective effects and can potentially modulate joint pain, and are, therefore, uniquely suited as potential disease-modifying osteoarthritis drugs.

The design and synthesis of substituted biphenyl libraries

A novel scaffold system for the generation of diversity libraries has been designed which allows for rapid modification not only of functional groups, but their spatial arrangements as well. The biphenyl scaffold allows for display of three or four diverse functional groups in a wide variety of spatial arrangements depending on the substitution pattern selected. The libraries are generated by a combination of solution and solid-phase chemistries and are cleaved off the solid-support for screening.

New developments in solid phase synthesis supports

Developments in solid phase synthesis supports for the end of 1996 and much of 1997 are reviewed. Issues regarding support induced impurities and enhancing solid phase reaction rates are also discussed.

Novel magnetic supports for small molecule affinity capture of proteins for use in proteomics

Magnetic supports are tested for use in batch affinity capture of proteins. Two types of magnetic polymer composites were used for solid phase synthesis and for the batch affinity chromatography of folate binding protein from a protein mixture. Gly-Gly-L-Methotrexate as well as other analogs were synthesized on magnetic supports consisting of either polyoxyalkyleneamine grafted onto polystyrene beads or a copolymer of polyethylene glycol dimethylacrylamide (PEGA). Both supports incorporated within their matrix sub-micron particles of paramagnetic magnetite. The peptide-methotrexate analogs were attached to the magnetic supports via a photocleavable linker. The bound methotrexate-peptide analogs were equilibrated with a protein mixture consisting of bovine albumin, chicken albumin, folate binding protein, lysozyme, lactoferrin and lactoperoxidase precursor in phosphate buffered saline (PBS) and then after magnetically separating and washing the supports of any unbound components the bound protein was removed either through the photocleavage of the tethered methotrexate-peptide ligand or via exchange with soluble methotrexate. In all cases, the photocleavage or exchange with soluble methotrexate released folate binding protein as the major affinity captured protein. Of the two magnetic supports tested, the PEGA based support was found to be superior to the polyoxyalkyleneamine grafted polystyrene support and comparable to beaded agarose in releasing bound folate binding protein. Of the two methods for removing bound protein, photocleavage of the covalently attached ligand was found to release exclusively folate binding protein as opposed to exchange with soluble methotrexate which released residual amounts of the non-specifically bound proteins bovine and chicken albumin, in addition to folate binding protein. Thus, use of the PEGA based magnetic support in conjunction with a photocleavable linker should help facilitate the automation of multipleparallel affinity chromatography for proteomics applications.

Use of silica gels and mesoporous molecular sieves as supports for the solid-phase Claisen rearrangement.

A series of silica gels and mesoporous molecular sieves differing in both the range of particle size and mean pore size were derivatized with the p-[(R,S)-α-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-p henoxyacetic acid linker and their loading capacities were measured. Loading capacities ranging between 0.4–0.6 mmol Fmoc/g were achieved. Several of these silica based materials were derivatized with the hydroxymethyl benzoic acid linker and used as supports for the solid phase Claisen rearrangement of a support bound phenyl allyl ether. Both the silica gel and mesoporous supports were heated at 225 °C for 3 h to effect the Claisen rearrangement. The results showed that, compared to the same reaction run homogeneously, the silica gel support achieved similar total product yields and ratios for two Claisen products. The mesoporous supports were found to selectively produce one of the Claisen products over the other. Analysis shows that the molecules bound to the mesoporous support are physically further separated from each other as compared to those bound to the silica gel support. A mechanism is presented which accounts for the selectivity of the mesoporous support in forming one Claisen product over the other. The Claisen product was further derivatized to the resulting phenyl ethyl ether through a solid phase Mitsunobu reaction on the mesoporous support.

Selection of supports for solid-phase organic synthesis

The field of peptide synthesis has been one of the engines that have driven the development of new supports and linkers for solid-phase organic synthesis. Pioneering work by Merrifield and others in peptide synthesis has made 1% cross-linked polystyrene the standard support for solid-phase organic chemical reactions [1,2]. The synthesis of non-peptide molecules has placed demands on this support that, in some cases, have produced less-than-optimal results. There are several issues unique to the solid-phase production of non-peptide molecules that differentiate it from the area of peptide synthesis. This differentiation comes from the fact that a larger variety of different compounds are expected to be produced in a combinatorial library and hence this translates to a larger number of different reactions that are attempted. This is in contrast to standard solid-phase peptide synthesis where one is dealing with a set number of reaction types (i.e., base or acid deprotection, amide coupling, peptide cleavage). Therefore, several important issues must be considered before choosing a support for combinatorial library production, such as whether the support is compatible with the intended reaction conditions and/or reaction solvent. Even if that does not pose a problem, one will find that transferring a reaction from solution to a solid support can dramatically affect the reaction rate. Additionally, there is the issue of how to monitor the solid-phase reaction. The type of support one chooses can sometimes restrict the type of method one uses to monitor the solid-phase reaction. Lastly, there is the issue of the loading capacity of the support, which is proving to be a far more important issue for the field of non-peptide combinatorial chemistry than, for example, the fields of solid-phase peptide or oligonucleotide synthesis. These are the issues that will be dealt with in the following review and, like in many things, one will find that achieving the ideal in one area will, in many instances, be a result of compromises in other areas.