MMP-9 (IC50 = 5 nM); MMP-9 (IC50 = 0.26 nM); MMP-2 (Ki = 0.05 nM); MMP-1 (IC50 = 79 nM); MMP-3 (IC50 = 6.3 nM); MMP-3 (Ki = 0.3 nM); collagenases 3 (Ki = 0.03 nM)

Prinomastat (AG3340; 0.1–1 µg/mL; 4 days; C57MG/Wnt1 cells) suppresses the production of MMP-3 that is induced by Wnt1. Wnt1-induced EMT and β-catenin transcriptional activity are reversed by prinomastat [1]. Topflash activity in CT7 cells is increased when L/Wnt3a cells and CT7 cells are co-cultured; similarly, co-culturing L/Wnt3a cells and MMP-3 overexpressing C57MG cells with CT7 cells increases Topflash luciferase activity in CT7 cells beyond the levels that are observed. Prinomastat (AG3340) inhibits these effects. The reduction of phosphorylated expression of Erk1/2 and cyclin D1 is correlated with Prinomastat's inhibition of C57MG/Wnt1 cells' S phase entry. Using an in vitro wound assay, the impact of Prinomastat on Wnt1-induced migration was then investigated. C57MG/Wnt1 cells migrated 1.8 times more than C57MG cells, as predicted. Prinomastat counteracts Wnt1's effects on vimentin's cellular distribution in C57MG/Wnt1 cells [1].

In the mouse model for human fibrosarcoma (HT1080), animals were injected intraperitoneally with 50 mg/kg/day for 14–16 days, beginning on day 3 and ending on day 6 following tumor inoculation. The animals responded well to Primastat, and there was no sign of weight loss or any side effects. Pronostat has a brief half-life of 1.6 hours and a good tumor growth inhibitory effect [1].

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Researchers studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.[3]

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The average proliferative vitreoretinopathy (PVR) scores in the prinomastat treatment and control groups were 2.62 and 3.57 respectively (p = 0.038; Wilcoxon rank sum). Clinically significant PVR with retinal detachment (PVR > or = grade 3) developed in 76% of rabbits in the control group versus 51% of rabbits treated with prinomastat. Conclusions: Intravitreally administered prinomastat decreased development of PVR in an experimental model which made use of dispase to induce PVR [4].

The design, synthesis, and structure-activity relationship (SAR) of a series of novel nonpeptidic cyclic phosphon- and phosphinamide-based hydroxamic acids as inhibitors of matrix metalloproteinases MMP-1, MMP-3, and MMP-9 are presented. Based on modelling studies and X-ray analysis, a model of the binding mode of these novel compounds in the MMP active site was obtained. This model provided a rational explanation for the observed SAR data, which included a systematic study of different S1' directed substituents, zinc-complexing groups, chirality, and variation of the cyclic phosphon- and phosphinamide rings. The in vivo effect of four compounds in a human fibrosarcoma mouse model (HT1080) was evaluated and compared to that of a reference compound, Prinomastat. Inhibition of tumour growth was observed for all four compounds[1].

Western Blot Analysis[1]
Cell Types: C57MG/Wnt1 Cell
Tested Concentrations: 0.1 µg/mL, 1 µg/mL
Incubation Duration: 4 days
Experimental Results: MMP-3 promoter activity was Dramatically diminished in C57MG/Wnt1 cells.

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Cell proliferation and cell motility assays. [2]
For FACS® analysis, cells were seeded into 100-mm plates and cultured to confluence in the presence or absence of 10 µg/ml Prinomastat (AG3340). Cells were washed in PBS, resuspended in 70% ethanol and stored overnight at 4°C. Cells were incubated with 20 µg/ml RNase A for 30 min at 37°C, centrifuged, and resuspended in PBS containing 40 µg/ml propidium iodide. The analysis was performed on an EPICS Elite ESP cell sorter using the Expo 32 (version 1.2) software. To assess cell migration, confluent monolayers of C57MG or C57MG/Wnt1 cells were scratch-wounded with a pipette tip to create a cell-free area. Cells were incubated for 24 h in the presence or absence of MMP inhibitors, and wound closure reflecting cell migration was documented by photography using an Olympus IMT-2 microscope equipped with an Olympus DPII digital camera. For each condition, 10 microscopic fields were selected along the wound and cells that had migrated from the wound edge to the cell-free space were counted.

To determine the efficacy of Prinomastat (AG3340), a synthetic inhibitor of matrix metalloproteinase, in the treatment of experimental proliferative vitreoretinopathy (PVR) induced by intravitreal dispase injection.
\n\nOne eye each of 53 New Zealand white rabbits was injected in the vitreous cavity with 0.07 unit of dispase to induce PVR. One week after PVR induction, 53 rabbits were randomized (27:26) to receive 0.5 mg prinomastat or the vehicle of the drug (acidified water) intravitreally every two weeks. The scores of PVR severity (scale of 1-5) were graded to compare the prinomastat-treated animals with the control group. [4]\n

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\n\nTumor Biology: Human Xenograft Studies [3]
\nHuman colon, prostate, lung, gastric and murine melanoma studies were conducted in the Laboratory Animal Resource Center Studies using COLO-320DM cells were initiated by harvesting serially passaged tumors (≃ 500-1000 mm3) from donor athymic mice and preparing 1- to 2-mm pieces for implantation into naive mice. Tumor cells or pieces were implanted bilaterally (2 sites/mouse). Studies using other human cell lines were initiated by harvesting exponentially growing cells from cell culture and preparing suspensions for s.c. implantation. Mice were randomized, ear-punched for identification, and housed in groups of 3/cage after tumor implantation; each study consisted of control and Prinomastat (AG3340)-treated groups containing 10-12 animals/group. Tumors were generally allowed to establish for 5 days prior to beginning dosing with AG3340 or vehicle (sterile water, pH 2.3). AG3340 was administered orally using sterile 20 g × 1.5 in. intragastric feeding needles. Animals were dosed 7 days/week, b.i.d., at approximately 9 am and 4 pm. Therefore, the total daily dose for a group given 100 mg/kg AG3340, b.i.d., was 200 mg/kg.\n
\nTumor growth was assessed by calculating volumes after measuring the length and width of subcutaneous tumors with electronic calipers. Volumes were calculated using the formula 1/2 (length)(width) 2. Additionally, effects of dosing regimens, vehicles and AG3340 on body weights and general health of mice were assessed throughout experiments.\n

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\n\nStudies Conducted at the University of Calgary [3]
\nHuman U87 glioma studies were conducted under approval by the Institutional Animal Care and Use Committee. Studies were initiated by implanting 5 × 106 cells/site in control and Prinomastat (AG3340)-treated groups containing 5-8 animals/group. Tumors were allowed to establish for 2 to 4 weeks before i.p. dosing with vehicle or Prinomastat (AG3340) began. Animals were dosed once daily, 5 days/wk (M-F); on Saturday, a double dose was given, and no dose was given on Sunday. Tumor areas were measured according to the area formula (length) × (width).\n

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\n\nStudies Conducted at Hoffmann-La Roche, Inc. [3]
\nHuman MDA-MB-435 breast cancer studies were conducted. Studies were initiated by implanting 1.5 × 106 cells in the mammary fat pad of athymic mice in control and Prinomastat (AG3340)-treated groups (n= 10/group). Tumors were allowed to establish for > 2 weeks before oral b.i.d. dosing with vehicle or Prinomastat (AG3340) began on a regimen of 7 days dosing/week. Tumor volumes were measured using the formula 1/2 (length)(width) 2.\n

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\n\nTumor Biology: Murine Intravenously Induced Metastasis Study [3]
\nB16-F10 cells in log-phase were detached from culture with 0.25% trypsin-EDTA, rinsed with media containing FCS, centrifuged, and resuspended in media without serum. Cells were then placed on ice. Experiments were conducted by randomizing control and Prinomastat (AG3340)-treated animals into 12 animals/group. 105 B16-F10 cells were implanted in 0.2-ml tumor into the tail-vein of C57BL/6 mice using 1-ml sterile syringes fitted with 30-g sterile needles. Cellular viabilities exceeded 90% and viabilities were maintained over the 1-2 hr period required to implant tumor cells into mice.\n

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\n\nEffect of MMP inhibition on tumor formation. [2]
\nMMTV-Wnt1 mice were bred and observed for mammary tumor formation in comparison to littermates receiving intra peritoneal injections at a dose of 50 mg/kg of Prinomastat (AG3340) twice a week. The mice were monitored for the development of a small tumor nodule by weekly palpation and the age at tumor onset was recorded.

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Preventive Treatment Study in MMTV-Wnt1 Transgenic Mice:** Female MMTV-Wnt1 mice (which develop mammary tumors due to Wnt1 overexpression driven by the MMTV promoter) were used. From 6 weeks of age, mice received intraperitoneal injections of AG3340 at a dose of 50 mg/kg twice weekly. Control littermates remained untreated. Mice were monitored weekly by palpation for the development of mammary tumors. The age at tumor onset was recorded, and Kaplan-Meier analysis was used to compare the tumor-free survival between treated and untreated groups [2].

Biological Half-Life
2-5 hours

[1]. Cyclic phosphinamides and phosphonamides, novel series of potent matrix metalloproteinase inhibitors with antitumour activity. Bioorg Med Chem. 2003 Dec 1;11(24):5461-84.

[2]. Stromelysin-1 (MMP-3) is a target and a regulator of Wnt1-induced epithelial-mesenchymal transition (EMT). Cancer Biol Ther. 2010 Jul 15;10(2):198-208.

[3]. Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials. Ann N Y Acad Sci. 1999 Jun 30;878:236-70.

[4]. The effect of prinomastat (AG3340), a potent inhibitor of matrix metalloproteinases, on a subacute model of proliferative vitreoretinopathy. Curr Eye Res. 2000 Jun;20(6):447-53.

Prinomastat is a hydroxamic acid with the chemical name (3S)-N-hydroxy-2,2-dimethylthiomorpholine-3-carboxamide, in which the hydrogen atom on the nitrogen atom of thiomorpholine is replaced by a [4-(pyridin-4-oxy)phenyl]sulfonyl group. It is a selective inhibitor of matrix metalloproteinases (MMPs) 2, 3, 9, 13, and 14. It possesses antitumor activity and can be used as an inhibitor of MMPs and EC 3.4.24.35 (gelatinase B). Prinomastat belongs to the class of hydroxamic acids, thiomorpholines, sulfonamides, aromatic ethers, and pyridines. It is the conjugate base of Prinomastat (1+). Prinomastat is a synthetic hydroxamic acid derivative with potential antitumor activity. Prinomastat inhibits angiogenesis, tumor growth, invasion, and metastasis by inducing extracellular matrix degradation through inhibition of matrix metalloproteinases (MMPs), particularly MMP-2, 9, 13, and 14. As a lipophilic drug, Prinomastat can cross the blood-brain barrier. (NCI04)

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Drug Indications
It has been investigated for the treatment of brain cancer, lung cancer, and prostate cancer.

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Matrix metalloproteinases (MMPs) play a well-defined role in the later stages of tumor progression. However, there is evidence that they are also involved in the early stages of malignant transformation. The Wnt signaling pathway plays a crucial role in the development and pathogenesis of many epithelial carcinomas. This study used Wnt1-induced epithelial-mesenchymal transition (EMT) in C57MG mouse mammary epithelial cells to investigate the role of MMPs in the early stages of malignant progression. Overexpression of Wnt1 in C57MG cells promoted EMT, translocation of β-catenin from the cell membrane to the nucleus and its transcriptional activity, cell proliferation, and cell migration. Meanwhile, we observed that compared with C57MG cells that did not express Wnt1, the expression of matrix metalloproteinase-3 (MMP-3) was increased in C57MG cells that expressed Wnt1, and the MMP-3 promoter activity was increased by 5.5-fold. After treating Wnt-overexpressing cells with the MMP inhibitor AG3340, the expression of MMP-3 was reduced. We also found that MMP-3 and Wnt3a synergistically enhanced the transcriptional activity of β-catenin in C57MG cells. Consistent with this, the effects of Wnt1 on EMT, proliferation and migration were inhibited after downregulation of MMP-3 by MMP inhibitors or siRNA. These results indicate that MMP-3 is both a direct transcriptional target of the Wnt/β-catenin signaling pathway and an essential component of this pathway. [2]

423.092

C, 51.05; H, 5.00; N, 9.92; O, 18.89; S, 15.14

192329-42-3

Prinomastat hydrochloride;1435779-45-5

466151

White to off-white solid powder

1.377g/cm3

1.615

4.123

2

8

5

28

638

1

O=C([C@@H]1N(S(=O)(C2=CC=C(OC3=CC=NC=C3)C=C2)=O)CCSC1(C)C)NO

YKPYIPVDTNNYCN-INIZCTEOSA-N

InChI=1S/C18H21N3O5S2/c1-18(2)16(17(22)20-23)21(11-12-27-18)28(24,25)15-5-3-13(4-6-15)26-14-7-9-19-10-8-14/h3-10,16,23H,11-12H2,1-2H3,(H,20,22)/t16-/m0/s1

(S)-N-hydroxy-2,2-dimethyl-4-((4-(pyridin-4-yloxy)phenyl)sulfonyl)thiomorpholine-3-carboxamide

AG3340; AG-3340; AG 3340; KBR-9896; CHEMBL75094; (3S)-N-hydroxy-2,2-dimethyl-4-(4-pyridin-4-yloxyphenyl)sulfonylthiomorpholine-3-carboxamide; 10T6626FRK;KBR 9896; KBR9896; Prinomastat.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :< 1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)