DNA polymerase alpha/delta; natural antiviral

Aphididicolin (0.5 μM, 5 μM; 0-5 d) exhibits modest cytotoxicity towards normal human embryonic cells, HeLa, H9, A549, and Caco-2 cell lines, but selectively kills neuroblastoma cells [4]. Aphidicolin, at 0.4 μg/mL, stops cell cycle progression in the G2 phase in 3 days [5]. Aphidicolin (1 μM; 24 hours) causes apoptosis in AtT-20 cells and reduces cell proliferation through the p53-GADD45β pathway at concentrations of 100 nM–10 μM [6]. Aphidicolin (10 μM; 0–6 hours) reduces Akt phosphorylation and (100 nM–10 μM; 24 hours) raises the mRNA levels of GADD45β, a putative p53 downstream target and a stress-responsive gene that causes growth arrest and DNA damage[6]. Varicella zoster virus (VZV) is inhibited by aphidicolin (10 μM; 0–6 h) with a low cytotoxicity and EC50 of 0.5–0.6 μM [7].

Aphidicolin glycinate (AG; NSC 303812), a reagent with a greater solubility, was used to create Aphidicolin. Aphidicolin Ganciate (100 mg/kg; i.p.; every 3 hours; 9 days) prolongs survival by up to 75% in mice with implanted B16 melanoma and M5076 sarcoma by acting as an antitumor [8].

---

Aphidicolin, an inhibitor of DNA polymerases alpha and delta, is cytotoxic in vitro against tumor cells. The poor solubility of aphidicolin has led to the development of aphidicolin glycinate (AG; NSC 303812), a water soluble ester currently in early clinical trials. The antitumor activity of AG was investigated in a series of transplantable murine tumors in vivo. The drug demonstrated activity against the i.p. implanted B16 melanoma, producing maximum increased life spans of 75% following i.p. administration every 3 h for three doses on days 1-9. Treatment schedules involving both single injections per day on days 1-9 and multiple injections per day on days 1, 5, and 9 were less effective, indicating that this antitumor activity is schedule dependent. Similarly, greater activity was observed against the i.p. M5076 sarcoma when three daily injections were given on days 1-9 (57% increased life span) than with a single injection either on days 1-9 (36% increased life span) or on days 1, 5, 9, and 13 (inactive). Further scheduling studies in the s.c. M5076 sarcoma model showed that a 7-day infusion was superior to both a 24-h infusion and a 7-day course of three bolus treatments per day. On the assumption that DNA polymerase inhibition is the basis for this antitumor activity, inhibition of DNA synthesis in BALB/c x DBA/2 F1 mice was investigated by measuring incorporation of [3H]thymidine (20 microCi, i.v.) into DNA of spleen and jejunum. At 2 h after administration of AG, inhibition of DNA synthesis was dose dependent (median inhibitory dose, 60 mg/kg in both tissues) and was > 99% at 300 mg/kg. The inhibition was rapid in onset; AG (100 mg/kg i.p.) produced maximal (> 98%) inhibition in both tissues at 30 min. Recovery occurred in the intestine within 16 h; in spleen recovery was delayed to 24 h, and was followed by a rebound incorporation at 48 h (203%). A comparison of the inhibition of thymidine incorporation in tumor cells (B16 melanoma and P388 leukemia) and normal jejunum revealed no significant differences in the extent of inhibition or the rapidity of recovery in these tissues. The rapid recovery of DNA synthesis inhibition supports the use of prolonged infusion schedules in clinical trials, but the lack of evidence of selectivity for tumor cells suggests that AG may be of limited therapeutic value as a single agent. Thus, we evaluated AG in combination with cisplatin in an in vivo model of cisplatin refractory human ovarian cancer[8].

Cytotoxicity assay [4]
Cell Types: NB
Cell Types: UKF-NB-1/2/3 and IMR-32
Tested Concentrations: 0.5 μM, 5 μM
Incubation Duration: 1, 2, 3, 4, 5 days
Experimental Results: Produced cells The enlargement and elongation of cellular processes before lysis occurs.

---

Cell cycle analysis [5]
Cell Types: Normal human diploid cells
Tested Concentrations: 0.4 μg/mL
Incubation Duration: 3 days or 7 days
Experimental Results: More than 80% of the cells entered the S phase and accumulated in the G2 phase. Completely inhibits cell growth without causing significant cell death.

---

Western Blot Analysis[6]
Cell Types: AtT-20 Cells Pituitary Adrenocorticotropin Tumor Cells
Tested Concentrations: 10 µM
Incubation Duration: 0 min, 5 min, 30 min, 2 hr, 6 hr, 24 hr
Experimental Results: AtT-20 Medium Phosphorylation of Akt was inhibited in cells within 5 min to 2 hr in a time-dependent manner. p27 protein levels increased between 30 minutes and 6 hrs (hours), and p53 protein levels increased Dramatically at 24 hrs (hours).

Animal/Disease Models: Mouse M5076 sarcoma subcutaneousmodel or B16 melanoma intraperitonealmodel [8]
Doses: 60 mg/kg, 100 mg/kg, 300 mg/kg
Route of Administration: intraperitoneal (ip) injection; once every 3 hrs (hrs (hours)); 9-day
Experimental Results: Dramatically inhibit tumor growth.

[1]. Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation. Biochemistry. 1988 Aug 23;27(17):6379-83.

[2]. Antiviral effects of aphidicolin, a new antibiotic produced by Cephalosporium aphidicola. Antimicrob Agents Chemother. 1973 Sep;4(3):294-8.

[3]. Aphidicolin potentiates apoptosis induced by arabinosyl nucleosides in human myeloid leukemiacell lines. Biochem Pharmacol. 1993 Dec 3;46(11):1909-16.

[4]. Aphidicolin selectively kills neuroblastoma cells in vitro. Cancer Lett. 1992 Dec 24;67(2-3):199-206.

[5]. Aphidicolin inhibits cell proliferation via the p53-GADD45β pathway in AtT-20 cells. Endocr J. 2015;62(7):645-54.

[6]. Fukuda M, Ohashi M. Aphidicolin inhibits cell growth by accumulation of G2 cells. Cell Biol Int Rep. 1983 Aug;7(8):579-85.

[7]. Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice. Antiviral Res. 2010 Jun;86(3):276-85.

[8]. Antitumor activity and biochemical effects of aphidicolin glycinate (NSC 303812) alone and in combination with cisplatin in vivo. Cancer Res. 1994 Feb 1;54(3):724-9.

Aphidicolin is a tetracyclic diterpenoid compound with the structure tetradecylhydro-8,11a-methylenecycloheptano[a]naphthalene, with two hydroxymethyl substituents at positions 4 and 9, two methyl substituents at positions 4 and 11b, and two hydroxyl substituents at positions 3 and 9. Aphidicolin is an antibiotic with antiviral and antimitotic activities. It is a reversible eukaryotic nuclear DNA replication inhibitor. Aphidicolin can function as an antibacterial agent, antiviral drug, antitumor drug, EC 2.7.7.7 (DNA-directed DNA polymerase) inhibitor, DNA synthesis inhibitor, apoptosis inducer, fungal metabolite, Aspergillus metabolite, and antimitotic agent. Aphidicolin is an antimitotic and antiviral metabolite of the aphid cephalosporium (Cephalosporium aphidicola). It inhibits DNA polymerase α and blocks DNA replication.
Aphidicolamin has been reported in Nigrospora oryzae, Pleospora bjoerlingii, and other organisms with relevant data.
Aphidicolamin is an antiviral antibiotic produced by Cephalosporium aphidicola and other fungi. It inhibits the growth of eukaryotic cells and certain animal viruses by selectively inhibiting the cellular replication of DNA polymerase II or virus-induced DNA polymerase. This drug can be used to control excessive cell proliferation in patients with cancer, psoriasis, or other dermatitis, with little or no adverse effects on non-proliferating cells.
Aphidicolamin is a novel antibiotic produced by Cephalosporium aphidicola. It is a potent inhibitor of cellular DNA synthesis and also strongly inhibits the growth of herpes simplex virus in tissue culture and rabbit eyes. Aphidicolamin is effective against iodide-resistant herpesviruses and does not readily induce drug-resistant strains of herpesviruses. [2] We investigated the effect of aphidicin, a DNA polymerase α and δ inhibitor, on apoptosis induced by arabinoside in the human promyelocytic leukemia cell line HL-60. Pretreatment of HL-60 cells with aphidicin (2 μM) significantly increased the number of morphologically apoptotic cells induced by 1-β-D-arabinofuranosylcytosine (ara-C) during a 4-hour incubation period. This was consistent with DNA fragmentation as quantified by diphenylamine or agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: this synergistic effect between aphidicin and arabinoside was also observed in other human myeloid leukemia cell lines U937 and KG-1. In addition, aphidicin enhanced apoptosis induced by 9β-D-arabinofuranosyladenine or 9β-D-arabinofuranosylguanine. [3] Aphidicin is a tetracyclic diterpenoid antibiotic known to inhibit the growth of eukaryotic cells by reversibly binding to DNA polymerase α, with no significant effect on cell viability in most common human cell lines. We observed that 5×10⁻⁷ M apigenin killed all cells in four human neuroblastoma cell lines. In contrast, apigenin had little effect on the viability of normal human embryonic cells and human continuum cell lines including HeLa, H9, A549 and Caco-2. Furthermore, in a medium co-cultured with normal embryonic cells, neuroblastoma cells were killed after treatment with 5×10⁻⁷ M apigenin, while normal embryonic cells continued to proliferate after the removal of apigenin. These results indicate that apigenin is a drug that selectively kills neuroblastoma cells in vitro. [4] Cushing's disease is mainly caused by pituitary adrenocorticotropic hormone (ACTH) adenomas that autonomously secrete ACTH. ACTH production may be related to tumor cell proliferation; however, the effect of cell cycle progression on ACTH production and cell proliferation in ACTH adenomas is unclear. Apigenin, a DNA polymerase inhibitor, can arrest cells at the S phase entry point and arrest the cell cycle; apigenin can also induce tumor cell apoptosis. In this study, we measured adrenocorticotropic hormone (ACTH) production and cell proliferation in AtT-20 adrenocorticotropic hormone tumor cells after apigenin treatment. Apigenin reduced the mRNA level of promelanocortinogen activating factor (POMC) in AtT-20 cells and the ACTH level in the culture medium for these cells. Apigenin also inhibited the proliferation of AtT-20 cells and induced apoptosis. Flow cytometry analysis showed that apigenin increased the proportion of cells in the G0/G1 phase and decreased the proportion of cells in the S phase. Apigenin reduced the phosphorylation levels of cyclic adenosine monophosphate response element-binding protein (cAMP) and Akt. Apigenin increased the levels of tumor proteins 27 (p27) and 53 (p53) while decreasing the level of cyclin E. Apigenin also increased the mRNA level of stress-reactive gene growth arrest and DNA damage-inducing protein 45β (GADD45β), a potential downstream target of p53. p53 knockdown led to an increase in GADD45β mRNA levels. GADD45β knockdown inhibited the reduction in cell proliferation. Therefore, apigenin inhibits the proliferation of AtT-20 cells through the p53-GADD45β pathway. [5]

---

To elucidate the in vivo targets of apigenin, we used flow cytometry to detect the cell cycle stages of normal human diploid cells under drug treatment. At drug doses that completely inhibited cell growth but did not show obvious cell death, more than 80% of cells entered the S phase and accumulated in the G2 phase. On the other hand, at higher doses, cells shed when they first entered the early S phase. Clearly, the in vivo effects of apigenin are far more complex than simple DNA synthesis inhibition. [6]

C20H34O4

338.4816

338.245

C, 70.97; H, 10.13; O, 18.91

38966-21-1

457964

White to off-white solid powder

1.2±0.1 g/cm3

507.8±50.0 °C at 760 mmHg

225-233ºC

230.4±24.7 °C

0.0±3.0 mmHg at 25°C

1.586

1.69

4

4

2

24

524

8

C[C@]12CC[C@H]([C@@]([C@@H]1CC[C@@H]3[C@@]24CC[C@@]([C@H](C3)C4)(CO)O)(C)CO)O

NOFOAYPPHIUXJR-APNQCZIXSA-N

InChI=1S/C20H34O4/c1-17(11-21)15-4-3-13-9-14-10-19(13,7-8-20(14,24)12-22)18(15,2)6-5-16(17)23/h13-16,21-24H,3-12H2,1-2H3/t13-,14+,15-,16+,17-,18-,19-,20-/m0/s1

(1S,2S,5R,6R,7R,10S,12R,13R)-6,13-bis(hydroxymethyl)-2,6-dimethyltetracyclo[10.3.1.01,10.02,7]hexadecane-5,13-diol

aphidicolin; 38966-21-1; (+)-Aphidicolin; NSC-234714; (+/-)-Aphidicolin; aphidocolin; ICI 69653; CCRIS 1783;

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 : ~50 mg/mL (~147.72 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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