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 synthesis of MMP-3 that is stimulated by Wnt1. Prinomastat reverses Wnt1-induced EMT and β-catenin transcriptional activity[1]. When L/Wnt3a and CT7 cells are co-cultured, the Topflash activity in CT7 cells increases. When L/Wnt3a and MMP-3 overexpressing C57MG cells are co-cultured with CT7 cells, the Topflash luciferase activity in CT7 cells increases beyond what is seen with L/Wnt3a cells. All of these effects are countered by Prinomastat (AG3340)[1]. When Prinomastat inhibits C57MG/Wnt1 cells' ability to enter the S phase, cyclin D1 expression and Erk1/2 phosphorylation both decrease. Next, utilizing an in vitro wound assay, the impact of Prinomastat on Wnt1-induced migration is investigated. As expected, relative to C57MG cells, the migration of C57MG/Wnt1 cells is boosted by 1.8 times. Prinomastat reverses Wnt1's influence on vimentin's cellular distribution in C57MG/Wnt1 cells[1].

In a mouse model of human fibrosarcoma (HT1080), mice are given 50 mg/kg/day ip daily commencing on day 3 to 6 following tumor inoculation, and this treatment is administered for 14–16 days. The animals handle prinomastat well, and there are no side effects or indications of weight loss. Prinomastat exhibits a short half-life of 1.6 hours and excellent tumor growth inhibition[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.

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 hydrochloride is the hydrochloride salt formed by reacting equimolar amounts of Prinomastat with hydrogen chloride. It is a selective inhibitor of matrix metalloproteinases (MMPs) 2, 3, 9, 13, and 14. It has a dual function as an MMP inhibitor, an antitumor drug, and an EC 3.4.24.35 (gelatinase B) inhibitor. It contains a Prinomastat (1+) domain. 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-yloxy)phenyl]sulfonyl group. It is a selective inhibitor of matrix metalloproteinases (MMPs) 2, 3, 9, 13, and 14. It has antitumor activity and can be used as an MMP inhibitor and an EC 3.4.24.35 (gelatinase B) inhibitor. It is a hydroxamic acid, belonging to the thiomorpholine, sulfonamide, aromatic ether, and pyridine classes. It is the conjugate base of prinostat (1+). Prinostat is a synthetic hydroxamic acid derivative with potential antitumor activity. Prinostat inhibits matrix metalloproteinases (MMPs) (especially MMP-2, 9, 13, and 14), thereby inducing extracellular matrix degradation and inhibiting angiogenesis, tumor growth, invasion, and metastasis. As a lipophilic drug, prinostat can cross the blood-brain barrier. (NCI04)

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Drug Indications
Investigations have been conducted in 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 cancers. 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 and enhanced the translocation and transcriptional activity of β-catenin from the cell membrane to the nucleus, as well as cell proliferation and migration. Simultaneously, we observed increased expression of matrix metalloproteinase-3 (MMP-3) and a 5.5-fold increase in MMP-3 promoter activity in Wnt1-expressing C57MG cells compared to C57MG cells. Treatment of cells overexpressing Wnt1 with the MMP inhibitor AG3340 reduced the expression of MMP-3. We also found that MMP-3 and Wnt3a synergistically enhanced the transcriptional activity of β-catenin in C57MG cells. Continued experiments showed that downregulation of MMP-3 by MMP inhibitors or siRNA could inhibit the effects of Wnt1 on EMT, proliferation and migration. 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]

C18H22CLN3O5S2

459.967381000519

459.068

C, 47.00; H, 4.82; Cl, 7.71; N, 9.14; O, 17.39; S, 13.94

1435779-45-5

Prinomastat;192329-42-3

10321991

White to off-white solid powder

3

8

5

29

638

1

Cl.S1CCN([C@@H](C(NO)=O)C1(C)C)S(C1C=CC(=CC=1)OC1C=CN=CC=1)(=O)=O

UQGWXXLNXBRNBU-NTISSMGPSA-N

InChI=1S/C18H21N3O5S2.ClH/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)1H/t16-/m0./s1

(S)-2,2-Dimethyl-4-((p-(4-pyridyloxy)phenyl)sulfonyl)-3-thiomorpholinecarbohydroxamic acid hydrochloride

Prinomastat HCl; AG3340; Prinomastat hydrochloride; 1435779-45-5; UNII-A8581Y0K6K; AG3340 hydrochloride; prinomastat HCl; AG-3340

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~100 mg/mL (~217.41 mM)
H2O : ~50 mg/mL (~108.70 mM)

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.)