Matrix Metalloproteinases

MMPs are a family of over 21 zinc-dependent neutral endopeptidases that play an important role in tumor angiogenesis, tissue remodeling, and cell migration.68-70 The two most important MMPs in cancer progression are MMP-2 (gelatinase A) and MMP-9 (gelatinase B).71,72 In cancer, levels of these MMPs are abnormally elevated, enabling cancer cells to degrade the extracellular matrix (ECM), invade the vascular basement membrane, and metastasize to distant sites.70

MMP targeting for tumor imaging or therapy can be achieved using small molecule inhibitors of MMPs, peptide sequences that target MMP, tissue inhibitors of MMPs, and MMP peptide substrates that are cleaved at the tumor site. For example, cyclic peptides containing a HWGF motif can specifically inhibit MMP-2 and MMP-9 activities and prevent both tumor growth and metastasis.73 Several small molecule MMP inhibitors such as the sulfonamide-hydroxamate compound CGS 27023A are currently in clinical trials.69,74-76

The protease functionality of MMP provides additional opportunities useful in enzyme prodrug therapy (Table 9.3). For example, drugs such as doxorubicin, auristatins, and duocarmycin may be conjugated to an acetyl-PLGL peptide sequence, MMP substrate, such that when incubated with MMP-2 or MMP-9, cleavage at the GL bond liberates a leucyl drug. Kline et al. showed that such a strategy produced no other cleavage intermediates and that proteolysis products were equipotent with the parent drugs.77

MMP-substrate-doxorubicin conjugates are found to be preferentially metabolized in murine fibrosarcoma xenografts relative to plasma and cardiac tissues.78 There was a 10-fold increase in

TABLE 9.3

Conjugates for Angiogenesis Targeted Drug Delivery

TABLE 9.3

Conjugates for Angiogenesis Targeted Drug Delivery

Compound

Drug

Target

Tumor Model

Reference

NGR-doxorubicin

Doxorubicin

Aminopeptidase N

MDA-MB-435 human mammary

64

carcinoma

mPEG-GPLGV-

Doxorubicin

MMP-2, MMP-9

Murine lewis lung carcinoma

79

doxorubicin

Albumin- GPLGIAGQ-

Doxorubicin

MMP-2, MMP-9

A375 human melanoma

82

doxorubicin

PLGL-doxorubicin

Doxorubicin

MMP-2, MMP-9

HT1080 human fibrosarcoma

78

RGD4C-doxorubicin

Doxorubicin

aVß3 integrin

MDA-MB-435 human mammary

64

carcinoma

RGD4C-doxorubicin

Doxorubicin

aVß3 integrin

MH134 murine hepatoma

132

RGD4C-AFK-doxorubicin

Doxorubicin

aVß3 integrin

HT1080 human fibrosarcoma

130, 131

E-c(RGDyK)2-paclitaxel

Paclitaxel

aVß3 integrin

MDA-MB-435 human mammary

202

carcinoma

PTK787-albumin-PEG-

PTK787 (kinase

aVß3 integrin

Human umbilical vein endothelial cells

21

c(RGDfK)

inhibitor)

c(RGDfC)-PEG-liposomes

5-FU

aVß3 integrin

Murine B16F10 melanoma

185

RGD-PEG-liposomes

Doxorubicin

aVß3 integrin

Murine B16F10 melanoma

203

c(RGDfC)-PEG-

Doxorubicin

aVß3 integrin

Cl-66 murine metastatic mammary

186

nanoparticles

tumor carcinoma

doxorubicin tumor-to-heart ratio and a greater effectiveness than free doxorubicin at reducing tumor growth. In particular, the conjugate cured 8 of 10 mice at levels below the maximum tolerated dose, whereas doxorubicin cured 2 of 20 mice at its maximum tolerated dose. Furthermore, mice treated with the conjugate had no detectable change in body weight or circulating reticulocytes.78

Other studies have focused on molecular modifications to modify in vivo biodistribution kinetics. For example, MMP substrate peptide-doxorubicin conjugates have been coupled to polyethylene glycol (PEG) to increase blood circulation time and to reduce non-specific cytotoxicity.79 The anti-cancer drug conjugate, mPEG-GPLGV-DOX, was synthesized by conjugating doxorubicin with GPLGV peptide (a substrate for MMP-2 and MMP-9) and PEG methyl ether (mPEG). In vivo experiments showed that a 50 mg/kg dose of mPEG-GPLGV-DOX had similar therapeutic effectiveness as a 10 mg/kg dose of doxorubicin, but it was associated with lower toxicity and prolonged life span relative to free drug.79

An interesting strategy to deliver drug therapy by EPR was investigated using a maleimide-doxorubicin conjugate of an octapeptide MMP substrate (GPLGIAGQ).80 The water-soluble substrate was designed to form an in situ complex with an albumin thiol (cysteine-34).81 The conjugate was efficiently cleaved by activated MMP-2 and MMP-9, liberating a doxorubicin tetrapeptide that showed antiproliferative activity in vitro in a murine renal cell carcinoma. When tested in a murine model of a human melanoma xenograft, the doxorubicin-octapeptide conjugate showed complete binding with albumin within 5 min and a 16 h stability half-life.82 The maximum tolerated dose of the conjugate was 4-fold greater than free doxorubicin. Furthermore, the conjugate showed superior anti-tumor effects as compared to free doxorubicin with duration of remission for up to 40 days and almost a 4-fold reduction in tumor size as compared to free doxorubicin at the end of the 50 day trial period.

Although imaging MMP expression is a relatively new area of research, a number of radiolabeled imaging agents have been tested in animal models (Table 9.2).19,20 I-125 labeled cyclic decapeptides containing HWGF have shown relatively disappointing results in mice bearing Lewis lung carcinoma.83 There was low to moderate uptake in tumor, significant tumor washout overtime, high concentrations in the liver and kidney, and low metabolic stability of the iodo-tyrosine complex.83 More promising results have been obtained with 18F and nC labeled MMP inhibitors developed for positron emission tomography (PET) imaging.84-86 In vitro studies showed no loss of MMP inhibitory activity of the radiolabeled compound as compared to the parent compound.84,87 Dynamic microPET images of tumor-bearing mice showed substantially higher tumor uptake than other background organs.88,89 The cyclic HWGF peptide has been conjugated with a 1,4,7,10-tetra-azacylcododecane-N,N/,N//,N///-tetraacetic acid (DOTA) chelate to enable 64Cu PET imaging.90,91 Studies of the 64Cu labeled peptide in a murine breast cancer model showed improved pharmacokinetics, tumor accumulation, and metabolic stability relative to a comparable 125I radiolabeled peptide.

111In-diethylenetriaminepentaacetic acid (DTPA)-N-TIMP-2, a radiolabeled high affinity endogenous tissue inhibitor of MMP, has been found to have similar activity as unmodified N-TIMP-2.92 Unfortunately, 111In-DTPA-N-TIMP-2 yielded disappointing results with insignificant tumor uptake in clinical studies in patients with Kaposi's sarcoma.93

Optical imaging methods to assess MMP activity has been achieved using a NIR fluorescence probe attached to a MMP-2 substrate.94-96 The MMP-2 imaging probe consists of three elements: a quenched NIR fluorochrome (Cy5.5), a MMP-2 peptide substrate (GPLGVRGK), and a PEG-poly-l-lysine (PEG-PLL) graft copolymer.94 The conjugate is designed such that the peptides are cleaved by MMP-2 at the tumor site, resulting in several hundred percent increases in the NIR fluorescence, allowing visualization of the MMP-2 in human fibrosarcoma tumor. Next MMP-2 inhibition was imaged after treatment with prinomastat where the treated tumor showed significantly lower NIR signal as compared to the untreated tumors.94 A similar NIR fluorescence-based probe has been designed for MMP-7 that is over expressed in colon, breast, and pancreatic cancer.97 In tumor extract containing MMP-7 in vitro, the fluorescence signal from the probe was enhanced 7-fold after enzymatic degradation. This probe could also be used to image tumor-associated enzyme activity and also inhibition of MMP-7 in vivo.

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