Aristolochic Acid and Chemically Modified Curcumin: an Overview

By Michael Baxter

ImageDr. Francis Johnson received his Ph. D. from Glasgow University in Scotland in 1954 and went on to become a postdoctoral fellow at Boston University until 1957. He worked at the Dow Eastern Research Laboratory before coming to Stony Brook University in 1973, where he became the founding vice-chairman of the Department of Pharmacological Sciences [1]. Currently Dr. Johnson is part of both, the Department of Chemistry and the Department of Pharmacological Sciences, and he is involved in studying the chemical aspect of genetic toxicology as well as organic synthesis and medicinal chemistry through small molecular drugs. Throughout the years, Dr. Francis Johnson has lent much of his attention to investigating both positive and negative characteristics of herbal remedies. In particular, he has focused on Aristolochia, which was used to induce labor in pregnant women, and curcumin, which is known to have anti-inflammatory properties as well as wound-healing capabilities. While working at Stony Brook University, Dr. Johnson became the co-founder and CEO of Chem-Master International, Inc. [2], a chemical synthesis company that aimed to resolve many of modern society’s medical problems, such as: periodontitis, inflammation, colon cancer, osteoporosis, arthritis, and other connective tissue deterioration conditions.

On the academic front, Dr. Francis Johnson works with a substance called aristolochic acid (AA), which translates into “best child birth” in Greek, a phytotoxin found in the vast genus of plants called Aristolochia, of which the most common species is Aristolochia clematitis. Women who were going into labor were induced by an herbal remedy containing aristolochic acid, however, little was known about the harmful effects of this herbal product until it went into phase I trials as an anti-tumor agent. A high frequency of nephropathy developed in patients and the herbal product was removed from clinical trials.

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AA (figure 1) is a potent and cumulative nephrotoxin that causes Aristolochic Acid Nephropathy (AAN), formerly known as Endemic Balkan Nephropathy (EN), due to its high prevalence in the Balkans region of Southeast Europe. In this area, Aristolochia clematitis grows in wheat fields, where it is accidentally incorporated into the flour made by the locals and subsequently ingested. AAN causes both kidney failure and upper urethelial cancer among patients who were exposed to AA.

Once AA is ingested, the liver reduces the AA nitro group into a nitronium ion, which is mutagenic. Normally, once a substance undergoes such a change, it becomes solubilized and it will travel to the kidneys and ultimately become excreted. However, AA accumulates in the kidneys and leads to tumors developing in the vicinity of the kidneys, most often in the upper ureter. The nitronium ion on the liver-processed AA can bind deoxy-adenine (dA) or deoxy-guanine (dG) through the terminal amino groups of each nucleobase. The aristolactam-DNA (AL-DNA) adduct that forms as a result of these interactions will bind itself into the DNA in a way that avoids detection by repair enzymes.

DNA damage occurs continually in our natural environment, which result in distortions or inversions. Repair enzymes can recognize these distortions and will remove the incorrect nucleotides, replacing them with matching ones. The human body will typically fix about 4,000 bits of DNA damage within a 24-hour period in each cell. However, DNA damage by AA goes unregulated and leads to carcinogenic mutation.

The covalent adduct, AL-DNA, tucks into the DNA helix and does not alter the DNA structure. As a result, repair enzymes do not recognize the modification made to that particular nucleotide as DNA damage and the lesion is preserved until RNA or DNA polymerases read the adduct as a random nucleotide and create mismatches during transcription and DNA replication, respectively. If cells noticed the DNA damage, various DNA repair mechanisms will be initiated, which if unsuccessful, can induce apoptosis to prevent proliferation of damaged DNA. Alternatively, these cells can accumulate irreversible and unfixable DNA damage, becoming undifferentiated epithelial rogue cells that reproduce uncontrollably in the upper ureter. Urethelial tumor cells were harvested from patients exposed to AA and were analyzed by Dr. Johnson and his colleagues. DNA damage in urethelial tumor cells was most consistently observed in the tumor suppressant gene TP53.

To isolate and study the damaged DNA, Dr. Johnson and colleges devised a novel method to produce completely synthetic damaged DNA that is chemically identical to AL-DNA produced in the urethelial cells of AAN patients. The de novo synthesis maintains complete control over where the lesion is in the DNA helix. Synthetic DNA is subsequently transformed into mice embryonic cells to observe how specific lesions behaved in vivo [4].

Various studies were carried out to determine the mechanisms behind the tumorigenicity of AA. It was determined that dA AL-DNA is significantly more mutagenic than dG AL-DNA, making the the dA adduct a more interesting research target (figure 2). However, both AL-DNA adducts result in the mismatched insertion of adenines, which leads to guanineàthymine and adenineàthymine transversions [3]. The mouse embryo model shows AL-DNA that is reincorporated into cells inhibits DNA synthesis, with the dA adduct showing more potency [4]. Furthermore, it was experimentally determined through 32P-postlabeling polyacrylamide gel electrophoresis (PAGE) that AA2 is slightly more carcinogenic than AA1 [5]. This study enabled Dr. Johnson and his colleagues to develop a quantitative measure of the amount of AL-DNA present in a cell, which will likely be adapted for diagnostic use in future drug models.

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Dr. Johnson, along with Stony Brook University and Chem-Master Int. Inc., also studies another herbal remedy called curcumin, a compound that gives saffron its golden color. Curcumin has many anecdotal, therapeutic uses that include its wound-healing, anti-inflammatory, anti-fungal and even anti-cancer effects. The critical problems with utilizing curcumin in human treatments are its insolubility in the bloodstream and its cytotoxicity at low concentrations [6].

Wound healing occurs in three distinct stages. The inflammatory phase normally occurs during the first 72 hours, when pro-inflammatory cytokines clot the wound. Immediately, the body recognizes the damage and signals for polymorphonuclear leukocytes, a type of white blood cell, to disinfect the wound and to protect against any foreign bacteria or viruses for the first 24 hours after the wound occurs. Macrophages arrive at the wound site about 48 hours after its formation and release high levels of matrix metalloproteinases (MMPs), specifically collagenase and gelatanase, to degrade all damaged collagen. MMPs continue to be secreted for up to 72 hours, in lower doses.

After the inflammatory phase, fibroblasts arrive and begin to generate new collagen, triggering the beginning of the collagen regeneration phase. Newly introduced collagen will be degraded by the remaining MMPs, however the fibroblasts will eventually overwhelm the MMPs, replacing all of the damaged collagen. Conversely, in diabetics fibroblast levels are insufficiently low, but the MMPs are persistent and continuously remove nascent collagen leading to chronic non-healing wounds.

A new drug, derived from curcumin, has now been developed that is much more effective at healing wounds. A functional assay was developed for chemically modified curcumin 2.24 (CMC2.24), in rats, to investigate its mechanisms in vivo. Diabetic rats were treated with CMC2.24 but were shown to conserve the levels of HbA1c, a protein found in greater concentrations in patients with diabetes, implying that the drug did not ameliorate the condition of diabetes.

A sample of fifteen rats was divided into five equal groups. Of those rats, twelve were injected with streptozotocin (STZ) to induce diabetes. The remaining three non-diabetic rats were used as a positive control for wound healing and did not receive any drugs. The diabetic rats were divided into four groups that received different concentrations of curcumin: 0%, a 1% topical solution, a 3% topical solution, and an oral dose of drug (30 mg/kg). Six standard sized wounds (six mm diameter) were made on the back of each rat. Results showed that, after seven days of treatment, the 1% topical solution of CMC2.24 healed wounds in diabetic rats with effectiveness comparable to what is seen in normal, non-diabetic rats that did not receive any drugs [6]. The oral solution of CMC2.24 was just as effective as the 1% topical solution of the drug, suggesting curcumin functions systemically as well as topically. Interestingly, the 3% topical solution of curcumin failed to match the effectiveness of the 1% topical solution (figure 3). These results suggest the mechanism of action of CMC2.24 inhibits MMP activity, but when the drug is present in high concentrations, it prevents the breakdown of damaged collagen, which ultimately impairs wound healing.

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In addition to reducing tissue loss and accelerating wound healing, CMC2.24 can be used to treat periodontitis, a disease involving tissue loss around the gums of teeth.CMC2.24 can also be used to reduce chronic inflammation in the joints of patients suffering from Arthritis by suppressing pro-inflammatory cytokines. Additionally, CMC2.24 is a novel type of drug, adding variety to treatment options that normally employ heavy doses of topical antibiotics, which, if misused, may lead to selective pressures that create drug resistant strains of various bacteria. However, the most important characteristic of CMC2.24 observed from current data is its lack of cytotoxicity, which enables researchers to safely administer the drug with minimal side effects. Finally, CMC2.24 has a relatively simple structure, is inexpensive to synthesize, and can be easily manufactured in large quantities at relatively low costs.

Dr. Francis Johnson has worked with his collaborators and Chem-Master Int. Inc. to expand the boundaries of drug discovery. During his career, Dr. Johnson helped design over 70 U.S. patents and has co-authored over 200 publications [1]. His studies include the field of genetic toxicology where he synthesizes damaged DNA to imitate the effects of the potent carcinogens. His current studies aim to improve our understanding of the oncological mechanism of AA by creating synthetic DNA with AL-DNA lesions incorporated at various locations in the DNA. While, it is unclear to researchers why AA specifically causes kidney failure, studies aim to elucidate the oncogenic mechanism of AA by chemically linking fluorophores to AA and determining the location of AA accumulation in kidneys. Dr. Johnson hopes to use this data to develop a practical, easily synthesizable drug with low cytotoxicity to treat AAN.

Dr. Johnson also studies CMC2.24, an easily synthesizable drug that inhibits inducible MMP enzymes, accelerating wound regeneration and inhibiting inflammation. While preliminary studies of CMC2.24 are promising, the drug requires additional research. The correct dosing for the topical ointment of CMC2.24 needs additional optimization and long-term effects of CMC2.24 are unknown. Research to elucidate long-term effects of CMC2.24 will require a considerable number of rats that will be exposed to topical and oral treatments of CMC2.24 for extended periods of time. If approved, CMC2.24 may eventually be used to accelerate wound healing, treat periodontitis, arthritis, and other inflammatory diseases.

Acknowledgements:

Francis Johnson, Arthur P. Grollman, Sivaprasad Attaluri, Radha Bonala, Kathleen Dickman, Mark Lukin, Charles Iden, Carlos De Los Santos, Lorne Golub, His-Ming Lee, Yu Zhang, Chem-Master Int. Inc.

References:

1)     DeRosa, Adam M. “Novel Therapeutic Matrix Metalloprotease And Pro-Inflammatory Cytokine Inhibitors.” Stony Brook University Research. Stony Brook State University of New York. Web. 2 Feb. 2012. <http://www.stonybrook.edu/research/otlir/technologies/Therapeutics/R-8134.pdf&gt;.

2)     Stony Brook University School of Medicine, Pharmacological Sciences, http://www.pharm.stonybrook.edu/news_events/news/francis-johnson-symposium-80-years-young.

3)     Bojan Jelaković, Sandra Karanović, Ivana Vuković-Lela, Frederick Miller, Karen L. Edwards, Jovan Nikolić, Karla Tomić, Neda Slade, Branko Brdar, Robert J Turesky, Želimir Stipančić, Damir Dittrich, Arthur P. Grollman and Kathleen G. Dickman. 2011. Aristolactam-DNA Adducts are a Biomarker of Environmental Exposure to Aristolochic Acid. International Society of Nephrology. 1523-1755.

4)     Sivaprasad Attaluri, Radha R. Bonala, In-Young Yang, Mark A. Lukin, Yujing Wen, Arthur P. Grollman, Masaaki Moriya, Charles R. Iden, and Francis Johnson. DNA Adducts of Aristolochic Acid II: Total Synthesis and Site-Specific Mutagenesis Studies in Mammalian Cells. Oxford Journals Vol. 38, No. 1 (2009): 339-52. Nucleic Acids Research. 23 Oct. 2009. Web. 15 Feb. 2012. <http://nar.oxfordjournals.org/content/38/1/339.full-text-lowres.pdf&gt;.

5)     Dong, Huan, Naomi Suzuki, Maria C. Torres, Radha R. Banala, Francis Johnson, Arthur P. Grollman, and Shinya Shibutani. Quantitative Determination of Aristolochic Acid-Derived DNA Adducts in Rats Using 32P-Postlabeling/Polyacrylamide Gel Electrophoresis Analysis. Drug Metabolism & Disposition Vol. 34.No. 7 (2006): 1122-127. Web. 24 Feb. 2012. <http://dmd.aspetjournals.org/content/34/7/1122.short&gt;.

6)     Lorne M. Golub, His Ming Lee, Francis Johnson. 2011. A Novel Chemically-Modified-Curcumin, CMC 2.24, Improves Wound-Healing Impaired by Diabetes.

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