In the early and late stages of 3T3-L1 preadipocyte differentiation, tafluprost acid (10, 100 nM) effectively inhibits adipogenesis[2]. In primary adipocytes from wild-type mice, tafluprost acid (100 nM) reduces adipogenesis, but not in those from FP knockout mice[2]. Human umbilical vascular endothelial cells (HUVECs) are stimulated to proliferate and migrate when exposed to tafluprost acid (10-4 M) for six hours [4]. HUVEC tube formation is stimulated (-4 M, 4–18 hours)[4].

The IOP-lowering effect of Taf/T-FDC was almost equivalent to that of Lat/T-FDC at 4-8 h after instillation. The peak IOP reduction of Taf/T-FDC and Lat/T-FDC was observed at 8 h after instillation, and there is no difference between the two. The difference between them was observed at 24-30 h after instillation, and Taf/T-FDC demonstrated a significantly greater IOP-lowering effect than Lat/T-FDC at 24-30 h after instillation. The IOP-lowering effect of Taf/T-FDC was sustained up to 30 h after instillation, while that of Lat/T-FDC had almost disappeared at 28 h after instillation. Timolol concentrations in aqueous humor after Taf/T-FDC instillation were higher than those after Lat/T-FDC instillation (Cmax, 3870 ng/mL vs 1330 ng/mL; AUCinf, 3970 ng·h/mL vs 1250 ng·h/mL). The concentrations of Tafluprost acid/AFP-172 and latanoprost acid in aqueous humor after instillation of Taf/T-FDC and Lat/T-FDC, respectively, were similar to those after instillation of mono-preparations of tafluprost and latanoprost, respectively. The cytotoxic effect of Taf/T-FDC to the human corneal epithelial cells was significantly lower than that of Lat/T-FDC at all evaluated time points in both undiluted and 10-fold diluted FDCs[3].

To evaluate the pharmacological characteristics of AFP-168 (tafluprost), a new prostaglandin (PG) F(2alpha) derivative, we examined its receptor-binding affinities, intraocular pressure (IOP)-lowering effect, effects on aqueous humor dynamics, and stimulating effect on melanogenesis. The receptor-binding profile for Tafluprost acid/AFP-172, a carboxylic acid of AFP-168, was determined by measuring muscle contractions in an organ bath, inhibition of platelet aggregation, and competitive binding of a radio-labelled ligand. For the IOP-measurement study, ocular normotensive and laser-induced ocular hypertensive cynomolgus monkeys were used, and IOP was measured using a pneumatonograph. For the studies of aqueous humor dynamics, IOP (Goldmann applanation tonometry), fluorophotometry, two-level constant pressure perfusion, and isotope dilution and accumulation techniques were used in ocular normotensive monkeys. The melanin contents in the medium and in the cell bodies of cultured B16-F0 melanoma cells were measured. The affinity for the FP receptor shown by Tafluprost acid/AFP-172 (Ki : 0.4 nm) was 12 times that of PhXA85 ( Ki : 4.7 nm), a carboxylic acid of latanoprost. A single application of AFP-168 at 0.0025% significantly lowered IOP in both ocular normotensive and hypertensive monkeys (3.1 and 11.8 mmHg, respectively, p < 0.01) and latanoprost at 0.005% significantly lowered IOP (2.1 mmHg, p < 0.01 and 9.5 mmHg, p = 0.059 respectively). Once daily instillation of AFP-168 at 0.001, 0.0025, or 0.005% for 5 days in normotensive monkeys significantly reduced IOP not only for a few hours, but also at the drug-trough time 24hr after application. Latanoprost at 0.005% also reduced IOP, but not at the drug-trough time. AFP-168 decreased IOP mainly by increasing uveoscleral outflow by 65% (p < 0.05) and, as sometimes seen with other prostanoids, also increased total outflow facility (33% increase, p < 0.05). In cultured B16-F0 melanoma cells, Tafluprost acid/AFP-172 (100 microM) did not stimulate melanogenesis, but PhXA85 (100 microM) did. These findings indicate that AFP-168 has a high affinity for the prostanoid FP receptor, has potent IOP-lowering effects in both ocular normotensive and hypertensive monkeys that exceed those of latanoprost, and has less stimulating effect on melanogenesis in melanoma cells[1].

The 3T3-L1 preadipocytes were treated to promote differentiation into mature adipocytes. During the early and late stages of differentiation (days 0, 2, and 7), 1 to 1000 nM latanoprost acid (LAT-A), travoprost acid (TRA-A), Tafluprost acid (TAF-A), bimatoprost (BIM), bimatoprost acid (BIM-A), unoprostone (UNO), or prostaglandin F2a (PGF2α) was applied to cells. Oil red O staining was used to detect intracellular lipids on day 10. Stained areas measured on a photograph were compared with those in control cultures. All experiments were performed in a masked manner. Next, similar experiments were performed using primary cultured mouse adipocytes from FP receptor knockout and wild-type mice. Results: When PGs were added on day 0 or 2, LAT-A, TAF-A, BIM-A, and PGF2α significantly inhibited adipogenesis (P < 0.01 on day 0, P < 0.05 on day 2) at concentrations of 10 nM and 100 nM, and TRA-A inhibited adipogenesis only at 100 nM. Bimatoprost and UNO did not affect adipogenesis at any concentration. When PGs were added on day 7, 100 nM LAT-A, BIM-A, or PGF2α significantly suppressed adipogenesis (P < 0.05). In mouse primary adipocyte cultures, LAT-A, TAF-A, BIM-A, TRA-A, and PGF2α significantly suppressed adipogenesis in wild-type adipocytes (P < 0.05), but adipogenesis was not suppressed by any of the PG compounds in FP knockout mouse adipocytes. Conclusions: Prostaglandin analogues have the potential to inhibit adipogenesis through FP receptor stimulation. Although these findings should be further analyzed in model systems more closely related to orbital fat, PG analogues may directly lead to reduced orbital fat by inhibiting adipogenesis[2].

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HUVECs cultured in the presence or absence of FP receptor antagonist (10 nM AL-8810) were exposed to escalating concentrations of 10(-7), 10(-6), 10(-5), 10(-4) and 10(-3) M Tafluprost acid/AFP-172 (the free acid of tafluprost). For cell proliferation assays, the numbers of cells were derived from a CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega) by Microplate reader. Endothelial cell migration was evaluated by a BD Biocoat™ Angiogenesis System using FluoroBlok ™ 24-well inserts. BioTek FLx800 fluorescence plate reader was used for quantitative measurement of fluorescently-labeled invasive vascular endothelial cells. Endothelial capillary-like tube formation was evaluated by BD Biocoat Angiogenesis System using Matrigel Matrix 96-well plate. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the gene expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and endothelial nitric oxide synthase (eNOS). COX-2 protein was detected by immunofluorescent staining and Western blot assay. Student's t-test was used for statistical analysis.[4]

The IOP-lowering effect of Taf/T-FDC and Lat/T-FDC in ocular normotensive monkeys was evaluated at 4 and 8 h after instillation in study A, at 12, 14, 16, and 18 h after instillation in study B, and at 24, 26, 28, and 30 h after instillation in study C. Drug penetration into the eye was evaluated by measuring the concentrations of timolol, Tafluprost acid/AFP-172 (active metabolic form of tafluprost), and latanoprost acid (active metabolic form of latanoprost) using liquid chromatography coupled with tandem mass spectrometry after single instillation of Taf/T-FDC or Lat/T-FDC to Sprague Dawley rats. Cytotoxicity following 1-30 min exposure of SV40-transformed human corneal epithelial cells to Taf/T-FDC or Lat/T-FDC was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays. Undiluted and 10-fold diluted solutions of each FDC were evaluated[3].

After infusion of Taf/T-FDC and tafluprost monotherapy, the concentration of tafluprost acid showed similar curves, reaching Cmax at 0.25–0.5 hours and decreasing with a half-life of 0.43–0.45 hours. After infusion of Lat/T-FDC and latanoprost monotherapy, the concentration of latanoprost acid also showed similar curves, reaching Cmax at 0.5 hours and decreasing with a half-life of 0.35–0.40 hours. [3]

[1]. Pharmacological characteristics of AFP-168 (tafluprost), a new prostanoid FP receptor agonist, as an ocular hypotensive drug. Exp Eye Res. 2004 Apr;78(4):767-76.

[2]. Activation of the prostanoid FP receptor inhibits adipogenesis leading to deepening of the upper eyelid sulcus in prostaglandin-associated periorbitopathy. Invest Ophthalmol Vis Sci. 2014 Mar 4;55(3):1269-76.

[3]. Advantages of Efficacy and Safety of Fixed-Dose Tafluprost/Timolol Combination Over Fixed-Dose Latanoprost/Timolol Combination. PLoS One. 2016 Jul 6;11(7):e0158797.

[4]. Effects of AFP-172 on COX-2-induced angiogenic activities on human umbilical vein endothelial cells. Graefes Arch Clin Exp Ophthalmol. 2012 Dec;250(12):1765-75.

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins. There are two isoenzymes of COX: COX-1 and COX-2. COX-1 is a housekeeping gene expressed in most tissues, while COX-2 is an inducible early intermediate gene induced in inflammatory cells, whose role is related to angiogenesis and tumorigenesis. In this study, 10⁻⁴ M of tafluprost acid/AFP-172 stimulated COX-2 expression in only one of the three genes. In human umbilical vein endothelial cells (HUVECs) pretreated with an FP receptor antagonist, tafluprost acid/AFP-172-induced COX-2 protein expression was blocked. These results suggest that tafluprost acid/AFP-172 induces COX-2 expression in HUVECs via the FP receptor, similar to the role of PGF2α in endometrial adenocarcinoma. Although COX-2's promotion of angiogenesis is well-known, the specific mechanisms by which COX-2-mediated angiogenesis participates in the process remain unclear. Unlike COX-1 inhibitors, COX-2 inhibitors are considered potential therapeutic agents for inhibiting angiogenesis and carcinogenesis. In this study, COX-2 inhibitors eliminated the pro-angiogenic effects of Tafluprost acid/AFP-172 by reducing the proliferation, migration, and tubular formation capacity of HUVECs. These results suggest that Tafluprost acid/AFP-172 promotes HUVEC angiogenesis through interaction with the COX-2 signaling pathway. COX-2 and VEGF appear to participate in angiogenesis through an interdependent dual gene expression pathway. Although COX-2 is known to regulate angiogenesis through interaction with the VEGF system, we found no difference in VEGF-A expression between the AFP-172-treated group and the control group using RT-PCR (Figure 5). Therefore, the pro-angiogenic effect of tafluprost/AFP-172 is independent of VEGF-A and eNOS. During ocular angiogenesis, hypoxia can stimulate angiogenesis through interaction with the VEGF, COX-2, and NOS systems. Thus, we can infer that the pro-angiogenic mechanism of tafluprost/AFP-172 does not depend on hypoxia-induced ocular angiogenesis, but rather on COX-2-mediated prostaglandin biosynthesis via FP receptors. In actual clinical applications, 10⁻⁴ M is far higher than the therapeutic concentration of tafluprost/AFP-172. Furthermore, the amount of eye drops that permeates from the cornea into the vitreous is approximately 1/104 of the dose reaching the vitreous. Since one drop of 0.0015% tafluprost eye drops contains approximately 2.5 μg of tafluprost, there may be 250 pg of tafluprost in the vitreous. Given the molecular weight of tafluprost (452.5), it is expected that 0.6 × 10⁻¹² M of tafluprost acid/AFP-172 can reach the vitreous cavity after a single clinical dose. Based on our observation that a concentration of 10⁻⁴ M of tafluprost acid/AFP-172 can stimulate angiogenesis in HUVEC cells, we hypothesize that therapeutic doses of tafluprost may not stimulate angiogenesis in glaucoma patients with anterior and posterior segment pathological angiogenesis diseases (such as neovascular glaucoma, keratitis, age-related macular degeneration (AMD), and diabetic retinopathy). However, in some respects, our in vitro experimental results cannot be directly applied to the treatment of patients, especially those with posterior segment angiogenesis diseases (such as AMD and diabetic retinopathy). First, ocular angiogenesis is associated with other ocular tissues, such as the retinal pigment epithelium and choroid, in addition to ocular endothelial cells. Secondly, although previous studies have explored ocular pathophysiology and pathogenesis using HUVEC cells, the similarity between HUVEC cells and ocular endothelial cells remains unclear. However, since a concentration of 10⁻⁴ M of Tafluprost acid/AFP-172 is still approximately 10,000 times higher than the pharmaceutical concentration of tafluprost (considering the therapeutic concentration of tafluprost eye drops is 0.6 × 10⁻⁸ M), tafluprost does not appear to stimulate anterior segment angiogenesis processes, such as neovascular glaucoma, corneal infection, and corneal transplantation. Our results are quite different from the angiogenic effects of latanoprost in a rat corneal model. This is likely due to the difference in the drug compound used and the experimental model. Tafluprost acid/AFP-172 is the active carboxylic acid form of tafluprost, and unlike latanoprost, tafluprost has two fluorine atoms at the 15-carbon position instead of a hydroxyl group. Our in vitro results may differ from in vivo results obtained in animal studies (e.g., the rat corneal angiogenesis assay used in latanoprost studies). In summary, we demonstrate that tafluprost acid/AFP-172 exerts its angiogenic effect by inducing COX-2 protein expression on HUVECs. For clinical applications, particularly in glaucoma patients with ocular neovascularization, further research, including in vivo studies, is needed to fully explore the angiogenic mechanism of tafluprost.

C22H28F2O5

410.46

410.19

C, 64.38; H, 6.88; F, 9.26; O, 19.49

209860-88-8

9978917

Colorless to light yellow ointment

1.3±0.1 g/cm3

575.9±50.0 °C at 760 mmHg

302.1±30.1 °C

0.0±1.7 mmHg at 25°C

1.579

2.8

3

7

11

29

557

4

C(=C/C[C@@H]1[C@@H](/C=C/C(COC2=CC=CC=C2)(F)F)[C@@H](C[C@@H]1O)O)/CCCC(=O)O

KIQXRQVVYTYYAZ-VKVYFNERSA-N

InChI=1S/C22H28F2O5/c23-22(24,15-29-16-8-4-3-5-9-16)13-12-18-17(19(25)14-20(18)26)10-6-1-2-7-11-21(27)28/h1,3-6,8-9,12-13,17-20,25-26H,2,7,10-11,14-15H2,(H,27,28)/b6-1-,13-12+/t17-,18-,19+,20-/m1/s1

(Z)-7-[(1R,2R,3R,5S)-2-[(E)-3,3-difluoro-4-phenoxybut-1-enyl]-3,5-dihydroxycyclopentyl]hept-5-enoic acid

UNII-WTV8EPZ396; Tafluprost acid; 209860-88-8; AFP-172; Tafluprost (free acid); WTV8EPZ396; UNII-WTV8EPZ396; 5-Heptenoic acid, 7-[(1R,2R,3R,5S)-2-[(1E)-3,3-difluoro-4-phenoxy-1-buten-1-yl]-3,5-dihydroxycyclopentyl]-, (5Z)-; 5-Heptenoic acid, 7-((1R,2R,3R,5S)-2-((1E)-3,3-difluoro-4-phenoxy-1-buten-1-yl)-3,5-dihydroxycyclopentyl)-, (5Z)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~243.64 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.)