Comparison of the pharmacology of hydroxamate- and carboxylate-based matrix metalloproteinase inhibitors (MMPIs) for the treatment of osteoarthritis.

OBJECTIVE AND DESIGN: Hydroxamic-and carboxylic-acid based matrix metalloproteinase inhibitors (MMPIs) were compared for their potency against various MMPs, pharmacodynamic properties and in vivo efficacy in a model of cartilage degeneration. MATERIALS AND METHODS: The MMPIs were evaluated for their ability to inhibit human MMPs using the quenched fluorescence assay. The ability of the MMPIs to inhibit the degeneration of the knee joint was evaluated in rats injected intraarticularly with iodoacetate. The amount of MMPI in the plasma and cartilage was determined using liquid chromatography/mass spectrometry/mass spectrometry (LC/ MS/MS). Plasma protein binding was measured by ultrafiltration and unbound MMPI was quantitated using HPLC. RESULTS: The hydroxamic acid based inhibitor PGE-3321996 and the carboxylic acids PGE-2909492 and PGE-6292544 were potent MMP-13 inhibitors, but only the hydroxamic acid PGE 3321996 demonstrated significant inhibition of knee degeneration in the rat iodoacetate model. Both of the carboxylic acids demonstrated superior pharmacokinetic properties and established much higher plasma concentrations than the hydroxamic acid. However, neither of the carboxylic acids was detectable in the cartilage, whereas, the hydroxamic acid was present in both the cartilage and the plasma. The carboxylic acid based MMPIs also demonstrated higher plasma protein binding (>99%) than the hydroxamic acid (79%). CONCLUSIONS: Carboxylic acid-based MMPIs were identified that had superior in vivo plasma exposure compared to a hydroxamic acid inhibitor but lacked in vivo efficacy in the rat iodoacetate model of cartilage degeneration. The lack of in vivo efficacy of the carboxylic acid based MMPIs were probably due to their lack of cartilage penetration which was related to their physicochemical properties.

Detection of aggrecanase- and MMP-generated catabolic neoepitopes in the rat iodoacetate model of cartilage degeneration.

OBJECTIVE: To characterize the time course of aggrecan and type II collagen degradation in the rat iodoacetate model of cartilage degeneration in relationship to the temporal sequence that has been described in human osteoarthritis (OA). DESIGN: Rats were injected intra-articularly in one knee joint with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The articular cartilage from the tibial plateau was harvested, extracted and glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay. Cartilage aggrecan neoepitopes were detected in cartilage extracts by Western blotting using antibodies recognizing the aggrecanase-generated C-terminal neoepitope NITEGE (BC-13) and the MMP-generated C-terminal neoepitope DIPEN (BC-4). A type II collagen collagenase-generated neoepitope was detected in cartilage extracts by ELISA using the Col2-3/4Cshort antibody; denatured collagen was detected using the Col2-3/4m antibody. RESULTS: Degenerative joint changes and proteoglycan (GAG) loss progressed with time after iodoacetate injection. Western blotting of cartilage extracts of iodoacetate treated rats demonstrated an increase in both aggrecanase- and MMP-generated epitopes with the NITEGE aggrecanase neoepitope being significantly elevated on days 7, 14 and 21 while DIPEN the MMP neoepitope was significantly elevated on days 7 and 14. The type II collagen neoepitope recognized by Col2-3/4Cshort was significantly increased in cartilage extracts of rats at days 14 and 21 after iodoacetate injection. CONCLUSION: The proteoglycan fragments extracted from the knee cartilage of rats after the intra-articular injection of iodoacetate appeared to result from cleavage at both aggrecanase and MMP sites. Cleavage of type II collagen by collagenase was also detected after iodoacetate injection and occurred subsequent to the initiation of aggrecan loss. These observations serve to demonstrate similarities in the mechanisms of cartilage degeneration induced by iodoacetate to those seen in articular cartilage in OA.

Induction of osteoarthritis in the rat by surgical tear of the meniscus: Inhibition of joint damage by a matrix metalloproteinase inhibitor.

OBJECTIVE: Characterize a model of osteoarthritis (OA) induced by a surgically transecting the medial collateral ligament and meniscus. Evaluate the effectiveness of a matrix metalloproteinase (MMP) inhibitor in this model. METHODS: The medial collateral ligament of the right knee of rats was transected and a single full thickness cut was made through meniscus. Rats were sacrificed at various times after the surgery to assess the severity of gross cartilage damage using an image analyser and microscopically by histology. The effect of an MMP inhibitor in this model was assessed by administering compound twice daily for the 21 days and evaluating gross and histological joint damage at day 21. The in vitro potency of the MMP inhibitor (MMPI) against a panel of human recombinant MMPs was assessed kinetically using a quenched fluorescent substrate. RESULTS: Surgical transection of the medial collateral ligament and meniscus resulted in a time dependent increase in the severity of the cartilage lesion (depth) as measured histologically but only a slight increase in the area of the lesion as assessed grossly by image analysis. Administration of a MMPI orally twice daily (b.i.d.) at 25mg/kg to rats in the meniscal tear model resulted in significant inhibition of cartilage degradation and osteophyte formation (total joint score) of 39+/-7% (mean+/-S.E.M., from four separate experiments). CONCLUSION: These results demonstrate that MMP inhibition is effective in reducing the joint damage that occurs in the meniscal tear model of OA and support a potential therapeutic role for MMP inhibition in the treatment of human OA.

The development of new carboxylic acid-based MMP inhibitors derived from a cyclohexylglycine scaffold.

A series of carboxylic acids was prepared based on cyclohexylglycine scaffolds and tested for potency as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors such as compound 18 display low nanomolar potency for MMP-2 and MMP-13, while selectively sparing MMP-1 and MMP-7.

Heterocycle-based MMP inhibitors with P2' substituents.

Potent and selective inhibition of matrix metalloproteinases was demonstrated for a series of sulfonamide-based hydroxamic acids. The design of the heterocyclic sulfonamides incorporates a six- or seven-member central ring with a P2' substituent that can be modified. Binding interactions of this substituent at the S2' site are believed to contribute to high inhibitory potency against stromelysin, collagenase-3 and gelatinases A and B, and to provide selectivity against collagenase-1 and matrilysin. An X-ray structure of a stromelysin inhibitor complex was obtained to provide insights into the SAR and selectivity trends observed for the series.

Development of new carboxylic acid-based MMP inhibitors derived from functionalized propargylglycines.

A series of carboxylic acids were prepared from a propargylglycine scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for four enzymes within the MMP family. The inhibitors were typically potent against collagenase-3 (MMP-13) and gelatinase A (MMP-2), while they spared collagenase-1 (MMP-1) and only moderately inhibited stromelysin (MMP-3). Compound 40 represents a typical inhibition profile of a compound with reasonable potency. Introduction of polar groups was required in order to generate inhibitors with acceptable water solubility, and this often resulted in a loss of potency as in compound 63. High serum protein binding proved to be a difficult hurdle with many compounds such as 48 showing >99% binding. Some compounds such as 64 displayed approximately 90% binding, but no reliable method was discovered for designing molecules with low protein binding. Finally, selected data regarding the pharmacokinetic behavior of these compounds is presented.

Moderation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors.

OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) inhibitors in mono-iodoacetate-induced arthritis in rats. DESIGN: The ability of compounds to inhibit MMPs in vitro was assessed kinetically using a quenched fluorescent substrate. Rats were injected with iodoacetate intraarticularly in one knee joint and damage to the tibial plateau was evaluated from digitized images captured using an image analyser and by histology. Collagenase and gelatinase activity in cartilage from iodoacetate injected knees were evaluated using(3)H-rat type I collagen and gelatin zymography, respectively. RESULTS: Collagenase and gelatinase activity significantly increased in the knee cartilage of rats injected with iodoacetate with peak activity by day 7. Three MMP inhibitors were examined for their efficacy in the rat iodoacetate-induced arthritis model. Significant (P< 0.05) inhibition of cartilage damage was observed in animals treated orally with 35 mg/kg b.i.d. of the three different MMP inhibitors. Inhibition of cartilage damage by the MMP inhibitors ranged from 36-42%. CONCLUSION: MMP inhibitors are partially protective against cartilage and subchondral bone damage induced by iodoacetate. These results support an important role for MMPs in mediating the joint damage in this model of arthritis.

Design and synthesis of piperazine-based matrix metalloproteinase inhibitors.

A new generation of cyclic matrix metalloproteinase (MMP) inhibitors derived from dl-piperazinecarboxylic acid has been described. The design involves: incorporation of hydroxamic acid as the bidentate chelating agent for catalytic Zn(2+), placement of a sulfonamide group at the 1N-position of the piperazine ring to fill the S1' pocket of the enzyme, and finally attachment of diverse functional groups at the 4N-position to optimize potency and peroral absorption. A unique combination of all three elements produced inhibitor 20 with high affinity for MMPs 1, 3, 9, and 13 (24, 18, 1.9, and 1.3 nM, respectively). X-ray crystallography data obtained for MMP-3 cocrystallized with 20 gave detailed information on key binding interactions defining an overall scaffold geometry for piperazine-based MMP inhibitors.

Development of new hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines.

A series of hydroxamates was prepared from an aminoproline scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors, such as compound 47, display broad-spectrum activity with sub-nanomolar potency for some enzymes. Modifications of the P1' portion of the molecule played a key role in affecting both potency and selectivity within the MMP family. Longer-chain aliphatic substituents in this region of the molecule tended to increase potency for MMP-3 and decrease potency for MMP-1, as exemplified by compounds 48-50, while aromatic substituents, as in compound 52, generated broad-spectrum inhibition. The data is rationalized based upon X-ray crystal data which is also presented. While the in vitro peroral absorption seemed to be less predictable, it tended to decrease with longer and more hydrophilic substituents. Finally, a rat model of osteoarthritis was used to evaluate the efficacy of these compounds, and a direct link was established between their pharmacokinetics and their in vivo efficacy.

Design, synthesis, and biological evaluation of potent thiazine- and thiazepine-based matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiazine- and thiazepine-based matrix metalloproteinase (MMP) inhibitors are described. The thiazine- and thiazepine-based inhibitors were discovered by optimization of hetererocyclic sulfonamide-based inhibitors. The most potent series of inhibitors was obtained by modification of the amino acid D-penicillamine. This amino acid provides a gem-dimethyl group on the thiazine or thiazepine ring which has a dramatic effect on the in vitro potency of this series. In particular, the sulfide 4a and the sulfone 5a were potent, broad-spectrum inhibitors of the MMPs with IC(50)'s against MMP-1 of 0.8 and 1.9 nM, respectively. The binding mode of this novel thiazepine-based series of MMP inhibitors was established based on X-ray crystallography of the complex of stromelysin and 4a.

Cartilage proteoglycan degradation by a mouse transformed macrophage cell line is mediated by macrophage metalloelastase.

OBJECTIVE AND DESIGN: Identify and characterize the matrix metalloproteinase responsible for cartilage proteoglycan degradation mediated by a macrophage cell line in a cell culture model that resembles some aspects of rheumatoid pannus. MATERIALS OR SUBJECTS: Supernatants from the transformed mouse macrophage cell line J774A.1 were used to purify the proteoglycan degrading activity. METHODS: J774A.1 macrophage culture supernatants were purified by sequential column chromatography and proteins were identified by zymography, western blotting and amino acid sequence analysis. Cartilage degradation was measured using 35S labeled bovine nasal cartilage. RESULTS: The cartilage degrading proteolytic activity in the mouse macrophage supernatants proved to be due to two major proteins with approximate molecular masses of 48 kDa and 22 kDa that were identified as macrophage metalloelastase (MME). Incubation of purified MME at 37 degrees C for up to 16 h resulted in the processing of the 48 kDa protein to several novel bands including a previously undescribed protein of approximately 25 kDa without accumulation of fully processed 22 kDa protein. A number of proteinases increased the rate of this processing. J774A.1 macrophage metalloelastase degraded cartilage proteoglycan with an efficiency approximately equal to human macrophage metalloelastase (MMP-12) and matrilysin (MMP-7) and twice that of stromelysin-1 (MMP-3). CONCLUSIONS: These data identify the cartilage proteoglycan degrading metalloproteinase secreted by J774A.1 macrophages in this cell culture model as MME, and describes mechanisms of activation and processing of this enzyme that may play an important role in cartilage degradation.

Design, synthesis, and biological evaluation of matrix metalloproteinase inhibitors derived from a modified proline scaffold.

The synthesis and structure-activity relationship (SAR) studies of a series of proline-based matrix metalloproteinase inhibitors are described. The data reveal a remarkable potency enhancement in those compounds that contain an sp(2) center at the C-4 carbon of the ring relative to similar, saturated compounds. This effect was noted in compounds that contained a functionalized oxime moiety or an exomethylene at C-4, and the potencies were typically <10 nM for MMP-3 and <100 nM for MMP-1. Comparisons were then made against compounds with similar functionality where the C-4 carbon was reduced to sp(3) hybridization and the effect was typically an order of magnitude loss in potency. A comparison of compounds 14 and 34 exemplifies this observation. An X-ray structure was obtained for a stromelysin-inhibitor complex which provided insights into the SAR and selectivity trends observed within the series. In vitro intestinal permeability data for many compounds was also accumulated.

Inhibition of human neutrophil elastase. 4. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of P2-modified, orally active peptidyl pentafluoroethyl ketones.

A series of P2-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE) are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluoroethyl ketone 1 as a model. Rational structural modifications were made at the P3, P2, and activating group (AG) portions of 1 based on structure-activity relationships (SAR) developed from in vitro (measured Ki) data and information provided by modeling studies that docked inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE) and subsequently the complex between 1 and HNE.

Inhibition of cartilage degradation in rat collagen-induced arthritis but not adjuvant arthritis by the neutrophil elastase inhibitor MDL 101,146.

OBJECTIVE AND DESIGN: The neutrophil elastase inhibitor MDL 101,146 was examined for its anti arthritic effect and to determine the role of neutrophil elastase in collagen-induced arthritis and adjuvant arthritis. MATERIAL: The collagen-induced arthritis model was performed in female DA rats immunized on day 0 an 1.7 with chick type II collagen in Freund's incomplete adjuvant. Adjuvant arthritis was induced in female Lewis rats by an intradermal injection of heat-killed Mycobacterium tuberculosis H37RA. METHODS: The clinical signs of arthritis were assessed, joint swelling was measured using calipers and bone degradation, new bone proliferation, pannus formation and cartilage degradation were evaluated histologically. RESULTS: MDL 101,146 administered orally inhibited the severity of collagen-induced arthritis as measured by a reduction in clinical score and joint swelling. Histological evaluation demonstrated a bone and cartilage sparing effect of MDL 101,146 in the tibio-tarsal joint of animals with collagen-induced arthritis. CONCLUSIONS: These results suggest that destruction of the joint in collagen-induced arthritis is at least partially due to neutrophil elastase.

Pharmacological evaluation of selected, orally active, peptidyl inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a serine proteinase capable of degrading a number of connective tissue macromolecules and has been implicated in the destructive processes associated with several chronic inflammatory diseases. A large series of peptidyl electrophilic ketones have been shown to be potent inhibitors of HNE in vitro and in vivo. We report the pharmacology and pharmacokinetics of selected inhibitors from this series. MDL 101, 146, MDL 102, 111, MDL 102,823 and MDL 100,948A are -Val-Pro-Val-pentafluoroethylketones with various amino-terminal protecting groups. Although their Ki values varied considerably, (25-170 nM), these compounds demonstrated similar ED50 values after oral administration in the HNE-induced hemorrhage model in hamsters and rats. The duration of action of MDL 102,111 was shorter than that of the other analogs in the HNE-induced pulmonary hemorrhage model in both species. The duration of action of all of the compounds was longer in the rat than in the hamster. Isolated sections of rat jejunum were used to determine the in situ absorption of these compounds. MDL 102,111 showed the greatest extent of absorption, with MDL 102,823, MDL 100,948A and MDL 101,146 following in descending rank order. The comparative metabolic stability of these analogs was measured over a 2-hr incubation period using rat liver homogenates. MDL 101,146 was the most stable, followed by MDL 102,823, MDL 102,111 and MDL 100,948A. MDL 101,146 was more stable in a liver homogenate from rats compared with a liver homogenate from hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human neutrophil elastase. 3. An orally active enol acetate prodrug.

Several analogs of N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N-[3,3,4,4,4-penta fluoro-1- (1-methylethyl)-2-oxobutyl]-L-prolinamide (1), in which the chiral center of the P1 residue has been eliminated, were synthesized and tested as inhibitors of human neutrophil elastase (HNE). Observations made during the course of this work led to the development of a single-step, stereoselective synthesis of E-enol acetate derivatives from HNE inhibitors containing a mixture of epimers at P1. In vitro studies, in the presence of added esterase, and 19F NMR studies, in biological media, indicated that the E-enol acetate derivatives should act as prodrugs in vivo. The ED50 value for (E)-N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N-[2- (acetyloxy)-3,3,4,4,4-pentafluoro-1-(1-methylethyl)-1-buteny l]-L-prolinamide (20), when administered orally in the hamster lung hemorrhage model, was 9 mg/kg.

Inhibition of human neutrophil elastase with peptidyl electrophilic ketones. 2. Orally active PG-Val-Pro-Val pentafluoroethyl ketones.

Valylprolyvalyl pentafluoroethyl ketones with different N-protecting groups were evaluated in vitro and in vivo as inhibitors of human neutrophil elastase (HNE). Several of these compounds were found to be orally active in HNE-induced rat and hamster lung hemorrhage models. The compound with 4-(4-morpholinylcarbonyl)benzoyl as the protecting group, 71 (MDL 101,146), was studied in greater detail. Hydration and epimerization studies were performed on 71 and related compounds in various media, including human blood serum. High-performance liquid chromatography studies on a reversed-phase system as a measure of the lipophilicity of 71 and related compounds revealed a small range of relative retention times wherein the orally active compounds fell. The Ki value determined for 71 vs HNE was 25 nM.

Inhibition of human neutrophil elastase and cathepsin G by a biphenyl disulfonic acid copolymer.

The sulfated polymer MDL 101,028 was found to be a potent-inhibitor of both human neutrophil elastase (HNE) and human neutrophil cathepsin G (CatG). Cleavage of synthetic substrate by HNE was inhibited by MDL 101,028 with an IC50 of 40 nM, while CatG was inhibited with an IC50 of 80 nM. Degradation of a macromolecular connective tissue substrate (cartilage proteoglycan) by HNE or CatG was inhibited by MDL 101,028 with an IC50 of approximately 10 microM. MDL 101,028 at concentrations of 4, 10 and 25 microM inhibited degradation of cartilage proteoglycan by human neutrophil lysate or stimulated human neutrophils by 54%, 70% and 79%, and 31%, 47% and 73%, respectively. Acute pulmonary injury resulting from the intratracheal (i.t.) instillation of HNE in rats was inhibited by 48%, 90% and 90% at concentrations of MDL 101,028 of 1.1 mg/kg, 2.8 mg/kg and 11 mg/kg. The duration of action of the compound after i.t. instillation was between 2 and 4 h. These results suggest that sulfated polymers such as MDL 101,146 may be useful as inhibitors of HNE-mediated lung injury.