Phenamil

Phenamil enhances the adipogenic differentiation of hen preadipocytes.[Pubmed:27460177]

Cell Biol Int. 2016 Oct;40(10):1123-8.

A study was conducted to examine the effect of Phenamil on adipogenic differentiation and expression of key adipogenic transcripts in hen preadipocytes. Preadipocytes were isolated from 20-week old Single Comb White Leghorn hens (Gallas gallus, Lohman strain). The experiment lasted for 48 h and had six treatments. Non-treated control (C) cells, cells treated with dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and oleic acid (DMIOA) (T1), DMIOA + 15 muM Phenamil (T2), DMIOA + 30 muM Phenamil (T3), 15 muM Phenamil alone (T4), and 30 muM Phenamil alone (T5). Neutral lipid accumulation and the mRNA expression of key adipogenic transcripts were measured in all treatments and compared. Lipid accumulation was detected in T1, T2, and T3 only. Expression of peroxisome proliferator receptor-activator gamma 2 (PPARgamma2), the core enhancer binding protein alpha (C/EBPalpha), C/EBPbeta, fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) as well as ETS variant 4 (ETV4) and 5 was higher (P < 0.05) in T2, T3, T4, and T5 compared to C. Expression of these transcripts was higher (P < 0.05) in T2 and T3 compared to T4 and T5. The core enhancer binding protein alpha, C/EBPbeta, and FABP4 were highly expressed (P < 0.05) in T1 compared to C. However, the expression of PPARgamma2, LPL, and ETV4 and ETV5 was not significantly different. Expression of C/EBPalpha, C/EBPbeta, and FABP4 was higher (P < 0.05) in T2 and T3 compared to T1. Expression of sterol regulatory element binding protein 1 (SREBP1) and leptin receptor (LEPR) was not significantly different among the treatments. In conclusion, Phenamil enhances DMIOA-induced adipogenic differentiation of hen preadipocytes but does not induce adipogenesis by itself.

Delivery of Phenamil Enhances BMP-2-Induced Osteogenic Differentiation of Adipose-Derived Stem Cells and Bone Formation in Calvarial Defects.[Pubmed:25869476]

Tissue Eng Part A. 2015 Jul;21(13-14):2053-65.

Bone morphogenetic proteins (BMPs) have been widely used for bone repair in the craniofacial region. However, its high dose requirement in clinical applications revealed adverse effects and inefficient bone formation, along with high cost. Here, we report a novel osteoinductive strategy to effectively complement the osteogenic activity of BMP-2 using Phenamil, a small molecule that can induce osteogenic differentiation via stimulation of BMP signaling. Treatment of adipose-derived stem cells (ASCs) with BMP-2 in combination with Phenamil significantly promoted the in vitro osteogenic differentiation of ASCs. The efficacy of the combination strategy of Phenamil+BMP-2 was further confirmed in a mouse calvarial defect model using scaffolds consisting of poly(lactic-co-glycolic acid) and apatite layer on their surfaces designed to slowly release Phenamil and BMP-2. Six weeks after implantation, the scaffolds treated with Phenamil+BMP-2 significantly promoted mouse calvarial regeneration as demonstrated by micro-computerized tomography and histology, compared with the groups treated with Phenamil or BMP-2 alone. Moreover, the combination treatment reduced the BMP-2 dose without compromising calvarial healing efficacy. These results suggest promising complementary therapeutic strategies for bone repair in more efficient and cost-effective manners.

Enhanced Mandibular Bone Repair by Combined Treatment of Bone Morphogenetic Protein 2 and Small-Molecule Phenamil.[Pubmed:27771997]

Tissue Eng Part A. 2017 Mar;23(5-6):195-207.

Growth factor-based therapeutics using bone morphogenetic protein 2 (BMP-2) presents a promising strategy to reconstruct craniofacial bone defects such as mandible. However, clinical applications require supraphysiological BMP doses that often increase inappropriate adipogenesis, resulting in well-documented, cyst-like bone formation. Here we reported a novel complementary strategy to enhance osteogenesis and mandibular bone repair by using small-molecule Phenamil that has been shown to be a strong activator of BMP signaling. Phenamil synergistically induced osteogenic differentiation of human bone marrow mesenchymal stem cells with BMP-2 while suppressing their adipogenic differentiation induced by BMP-2 in vitro. The observed pro-osteogenic and antiadipogenic activity of Phenamil was mediated by expression of tribbles homolog 3 (Trb3) that enhanced BMP-smad signaling and inhibited expression of peroxisome proliferator-activated receptor gamma (PPARgamma), a master regulator of adipogenesis. The synergistic effect of BMP-2+Phenamil on bone regeneration was further confirmed in a critical-sized rat mandibular bone defect by implanting polymer scaffolds designed to slowly release the therapeutic molecules. These findings indicate a new complementary osteoinductive strategy to improve clinical efficacy and safety of current BMP-based therapeutics.

Phenamil, an amiloride derivative, restricts long bone growth and alters keeled-sternum bone architecture in growing chickens.[Pubmed:28340021]

Poult Sci. 2017 Jul 1;96(7):2471-2479.

"Broiler-type" chickens are fast-grow-ing, heavy-bodied birds with high demands on bone quality. Phenamil increased mineralization in cultured murine mesenchymal stem cells. Phenamil effects were tested in 2 groups of weight and gender matched day-old broiler chickens (n = 13). Oral administration of 30 mg Phenamil/kg body weight d 1 to 13 reduced growth of chicks d 5 to 14 (P = 0.002); with Phenamil-treated (PT) chick body weight being 84% of vehicle-treated (VT) chicks' body weight on d 14. Tissues collected on d 15 showed that femur lengths and widths did not differ, but tibias from PT chicks were 6% shorter (P = 0.002) and 13% narrower (P = 0.012) with 18% thinner tibial cross-sections (P < 0.008) than in VT chicks. Angles of the caudal aspect of the anterior surface of keeled-sternums were 166 degrees in PT chicks, flatter than the 148 degrees found in VT chicks (P = 0.000). Total mineral content of both tibia and femur were lower in PT chicks (P = 0.005 for both). Bone Ca, P, and Mg (ppm) in ash were similar, but Ca:P was lower (1.70 vs 1.75) in PT versus VT chicks (P < 0.05). Osteocalcin was approximately 20% lower (P = 0.020), PINP was approximately 45% higher (P = 0.000) in PT chicks. Carboxy-terminal telopeptide type I collagen (ICTP) and cross-linked N-telopeptide of type I collagen (NTX1) were similar in the 2 groups. Phenamil had unexpected and detrimental effects on bone formation in growing broiler chicks, reducing linear skeletal growth and markedly changing bone architecture.

Inhibition of TRPP3 channel by amiloride and analogs.[Pubmed:17804601]

Mol Pharmacol. 2007 Dec;72(6):1576-85.

TRPP3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a Ca2+-activated channel permeable to Ca2+, Na+, and K+. TRPP3 has been implicated in sour tasting in bipolar cells of tongue and in regulation of pH-sensitive action potential in spinal cord neurons. TRPP3 is also present in excitable and nonexcitable cells of other tissues, including retina, brain, heart, testis, and kidney, with unknown functions. In this study, we examined the functional modulation of TRPP3 channel by amiloride and its analogs, known to inhibit several ion channels and transporters and respond to all taste stimuli, using Xenopus laevis oocyte expression, electrophysiology, and radiotracer measurements. We found that amiloride and its analogs inhibit TRPP3 channel activities with different affinities. Radiolabeled (45)Ca2+ uptake showed that TRPP3-mediated Ca2+ transport was inhibited by amiloride, Phenamil, benzamil, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Two-microelectrode voltage clamp experiments revealed that TRPP3-mediated Ca2+-activated currents are substantially inhibited by amiloride analogs, in an order of potency of Phenamil > benzamil > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 microM, respectively. The inhibition potency positively correlated with the size of inhibitors. Using cell-attached patch clamping, we showed that the amiloride analogs decrease the open probability and mean open time but have no effect on single-channel conductance. Study of inhibition by Phenamil in the presence of previously reported inhibitor tetrapentylammonium indicates that amiloride and organic cation inhibitors compete for binding the same site on TRPP3. TRPP3 may contribute to previously reported in vivo amiloride-sensitive cation transport.

Biochemical identification of two types of phenamil binding sites associated with amiloride-sensitive Na+ channels.[Pubmed:2546581]

Biochemistry. 1989 May 2;28(9):3744-9.

The existence of distinct forms of the epithelium Na+ channel that differ in their sensitivity to amiloride has been repeatedly suggested by physiological data. The biochemical basis for these differences was analyzed by using Phenamil, the most potent inhibitor known so far for the epithelium Na+ channel. [3H]Phenamil of high radioactive specific activity (30 Ci/mmol) was prepared and used to titrate [3H]Phenamil binding sites in pig kidney membranes. Kinetic experiments, equilibrium binding studies, and competition experiments indicated the presence in crude membrane preparations of two classes of independent binding sites. A first binding site was characterized by a high affinity for Phenamil (Kd1 = 0.4 nM) and for amiloride (Kd1 = 0.1 microM). A second binding site recognized Phenamil and amiloride with lower affinities [Kd2(Phenamil) = 28 nM, Kd2(amiloride) = 4 microM]. The ratio of the respective amounts of low- and high-affinity binding sites was 14 +/- 2 in different membrane preparations (range: 6-22). The two types of binding sites for [3H]Phenamil copurified and were still observed after purification of the epithelium Na+ channel to homogeneity. These results indicate that at least two types of pharmacologically distinguishable Na+ channels exist in the kidney. They correspond either to two isoforms of the apical Na+ channel or to one single type of channel under two different states of covalent regulation.