Gallium, key of the excellent Bamileke Artemisia?

Over the years IFBV-BELHERB accumulated puzzling data concerning Artemisia annua grown on the Bamileke plateau in Cameroon. Among all the clinical trials we have run in several countries, the infusion from Cameroon gave probably the best results (Rosine Chougouo et al, Proceedings MIM Conf, Nairobi, Kenya, 2 Nov 2009, no 312). The results of the comparative study showed a significantly higher sensistivity for the Artemisia annua concoction (0% late therapeutic failure), much better than 12.5 % for artesunate and 14.3% for artesunate-amodiaquine. This outstanding efficacy led us in 2010 to conduct a thorough analysis (monoterpenes, polyphenols, scopoletin, artemisinin) of 7 Artemisia annua samples from different villages in Cameroon, comparing them with samples from Belgium, Katanga, Luxembourg, Brazil, Central Africa, China, Senegal. At that stage the high artemisinin content of the Cameroon samples was the most plausible explanation. But we continued over the years accumulating the results of clinical trials with Artemisia annua plants with very low artemisinin contents which gave also excellent results (all results and data available on request). Some Artemisia species like absinthium, afra and apiacea containing no artemisinin are used with high antimalarial efficacy in many countries. We were puzzled in 2010 by a flow cytometry result obtained at Luxembourg : Artemisia annua from Luxembourg gave a lymphocyte activation by 17.8 % and the plant from Cameroon an insignificant 4.1% only. A thorough toxicity study of this plant had been made at the Université des Montagnes, Bangangte (G, Chuipet, PhD Thesis, 2011) and no acute nor chronic toxicity was evidenced. We were also puzzled by a result obtained at the University of Al Quds, Palestine, who published with us several papers on the beta-hematin inhibitory effect of medicinal plants like Artemisia sieberi, Salvia officinalis, Inula viscosa, Thymus vulgaris : the Artemisia annua from Cameroon shows a much stronger beta-hematin inhibition than the plant from Luxembourg. (M Akkawi, S Jaber…P Lutgen, MWJ 2014, 5,11). Any correlation with artemisinin is doubtful, as several authors claim that artemisinin does not inhibit hemozoin formation. And all attempts to relate the difference in inhibition efficiency to other molecules present in the two plants were fruitless. A further finding of our research at the University Al Quds is that sodium bicarbonate significantly enhances the beta-hematin inhibition of Artemisia sieberi (S Jaber, P Lutgen, M akkawi, submitted for publication) In 2012 we published a paper with the UCAD university of Dakar (T Alassane, M Diallo, P Lutgen et al.,) analyzing the element and mineral content of several herbs. In the Artemisia annua from Cameroon the content of several metals or elements: Fe, Mn, Al, Rb, Sr was twice as high as in the Artemisia annua from Luxembourg. This indicates that the plant in the North West of Cameroon is growing on a soil rich in minerals. And indeed the Bamiléké plateau in North West Cameroon has been extensively studied for its content in aluminium, iron but also gallium (B Hieronymus et al., J of Geochem Explor. 2001, 72, 147-163). In this bauxite rich soil gallium is found in concentrations around 50 mg/kg which compares with a common average of 10 mg/kg for other terrestrial soils. There is a selective uptake of gallium by plants : a higher ratio of Ga to Al is found in plants than in soils. The gallium concentration is around 1-10 ppm, 100 times higher than for other trace metals like As, Pb, Sr, Hg, Cd. In wheat gallium concentrations can go up to 2mg/g DM. Gallium also inhibits iron uptake, and Fe deficiency can be found in plants treated with gallium salts ( GV Johnson et al., Plant Physiol Biochem, 2007, 45-5, 302-8). Organic wastes from plant material, citrus peels, tea waste, olive pomace are used to remove gallium from aqueous solutions (Wie-Lung Chu et al., Fresenius Environmental Bulletin, 19-12, 2010, 2848-2856) Many plants of the Artemisia family are accumulators of metals and heavy metals. The concentration of certain metals may exceed the concentration which is toxic to other plants (E Alirzayeva et al. For. Snow Landsc Re 80, 3, 339-348). In Italy Artemisia absinthium and Artemisia campestris are often found on industrial sites contaminated by metals (N Massa et al., Ecotoxicology end Env Safety, 2010, 73, 1888-97). The stems and leaves of Artemisia tridentata are uranium acccumulators and indicators of uraniferous deposits in the western USA. Artemisia persia is a gold accumulator. Artemisia diffusa can carry remarkably high zinc levels. Gallium is a semimetallic element. Its biological actions stem from its ionic radius which is almost the same as that of the ferric iron, whereby it can replace iron in Fe(III)-dependant biological systems and enzymes. Unlike Fe(III), ionic Ga(III) cannot be reduced and when incorporated, it inactivates Fe(III) dependant oxidation and reduction processes, that are necessary for bacterial and mammalian cell proliferation. Incorporated in iron-specific enzymes and proteins it acts like a Trojan horse. Fe metabolism is of high vulnerability for infecting bacteria because they require Fe for growth. Restrictions in the ability to efficiently scavenge iron and use it usually lead to avirulence. Gallium is known to disrupt iron metabolism in Mycobacteria tuberculosis residing within human macrophages (O Olakanmi et al, Infection and Immunity, 2000, 5619-5627). It was found that gallium inhibits Pseudomonas aeruginosa growth and biofilm formation. 30% of nursing home patients are now resistant to existing drugs against this bacterium. Gallium porphyrins are potent antibacterial compounds. But their action is limited to bacteria expressing haem and hemoglobin utilization from the host and this includes most of the gram-positive, gram-negative and mycobacteria, including Straphylococcus aureus, Escherichiacoli, Vibrio cholerae, Shigella dysenteriae etc (I Stojiljkovic et al ., Molecular Microbiology, 1999, 31, 429-442). A key factor for this is that the infecting organisms build biofilms similar to polymer matrixes and a certain number of antibiotics even promote biofilm formation leading to chronic infection. It was possible to demonstrate (Y Kaneko et al., J Clin Investig, 117,4, 677-688) in batch cultures of Pseudomonas aeruginosa that the adddition of gallium nitrate blocked biofilm formation. Gallium nitrate is also used to prevent the formation of biofilms on medical devices (Yuanyuan Zhu et al., Exp and Therapeut Med., 2013, 5, 1001-4). Patent WO 2007053581 A2 describes a toothpaste where a gallium compound like nitrate or citrate has been added to fight microbial growth and dental caries. Extracellular polysaccharides of microbial origin are able to bind metals. Gallium readily formes complexes with polysaccharides like inulin and these complexes will improve bioavailability. Inulin-type fructans act as prebiotics and improve mineral absorption by their impact on the amelioration of gut health (K Scholz-Ahrens et al., The Journal of Nutrition, 2007). Limitation of Fe availability is utilized by many animal species as a means to host defense. Humans have evolved potent Fe-withholding or chelating mechanisms that can block acute infection. Gallium is highly effective in competing for the acquisition of iron. Most biological systems are unable to distinguish gallium from iron. Gallium nitrate and maltolate are FDA-approved drugs, the latter binding rapidly to proteins after oral intake Gallium compounds convert blood hypercalcemica into hypocalcemia, resulting in a marked reduction in urinary calcium. The radioactive isotope of gallium is used for scintigraphy imaging in inflammation and cancer. Ga(III) binds like Fe(III) to the human serum transferrin. Iron is paramagnetic and gallium is diamagnetic and by NMR spectroscopy it could be determined that the binding of the gallium ion to transferrin is strong (EL Dodd PhD thesis, McGill University 2009). Gallium accumulates in inflammatory lesions. Permeability indices for inflammatory tissue are much greater than for normal tissues. It stays there by binding to the acid mucopolysaccharide present in the tissue. Overproduction of TNF is considered a major pathogenic mechanism responsible for induction of fever in Plasmodium falciparum malaria. Gallium nitrates inhibit the release of inflammatory mediators like IL-6, reducing pain and injury (Min-Fu Tsan, The Journal of Nuclear Medicine, 26, 88-92. 1985). Gallium nitrate is very efficient in relieving and even eliminating arthritis pain (G Eby Med Hypotheses, 2005, 65 1136-41). Gallium nitraue also suppresses lupus eryththematosus. It increases the percentage of CD4 and CB8-bearing lymphocytes (G Apseloff et al. Naunyn Schmiedergs Arch Pharmacol 1997, 4, 517-25). We will not cover in this document the numeerous papers which describe the use of gallium nitrate or maltonate in cancer therapy. Gallium binds to transferrin in vivo. Ga-TF potently inhibits T lymphocyte cell activation. Lymphocytes like other cells require iron for cell division and by reducing the availability of iron gallium creates a limiting factor. The results obtained at Luxembourg in 2010 on lymphocyte activation with Artemisia annua from Cameroon can eventually be explained by this mechanism. Sodium bicarbonate plays an essential role in the binding of Fe(III) to transferrin (P Aisen et al., J Biol Chem, 1967, 242-10, 2484-92) and this ternary complex has been studied since 50 years. Transferrin is remarkable for both the strength and specificity of bicarbonate binding. Of all the anions tested, only those with structurally similar carboxyl groups were capable of substituting for bicarbonate. Why and how the addition of bicarbonate influences the beta-hematin inhibtion remains an open question. Gallium under physiological conditions does not enter erythrocytes, but it is possible that it has access to infected erythrocytes through new channels. A gallium based antimalarial drug had been developed in China in 1991 (G YAN et al, Acta Pharmacologica Sinica, 1991, 12, 530-33). The mode of action seems to be different from chloroquine. The therapeutical effect of gallium on Trypanosoma had already been noticed in 1931 (C Levaditi et al., CR Hebd Seances Acad Sci 1931). This tropical infection is also based on a hemoglobin-heme process. Gallium has solution- and coordination chemistries close to those of iron, it is able to replace iron in porphyrins, perturbing the ordered crystalline structure of hemozoin. It inhibits hemozoin formation attacking even chloroquine resistant parasites (V Sharma et al Chem Comm 1997, 2223-2224). It is believed that the pi-electron system of porphyrin rings leads to the formation of pi-pi adducts. These can be perturbed by gallium. The Artemisia annua from the Bamileke plateau is also very rich in aluminium: 23.40 mg/kg versus an average of 11 mg/kg for the other 7 tea samples analyzed. It is posssible that aluminium plays a role. The adjuvant activity of this metal was discovered more than 60 years ago. It decreases the heme biosynthesis in bone marrow cells of rats (K Zaman et al, Folia Haematol Int Blutforsch 1990, 117, 307-11). Aluminium chelation therapy in dialysis patients inhibits hemoglobin synthesis (P Altmann et al., Lancet, 1988, 1, 1012-5). Like many other metalloporphyrins it inhibits beta hematin formation (KA Cole et al., J Inorg Biochem, 2000, 78-2, 109-15). The in vitro antimalarial activity against Plasmodium falciparum and heme polymerization were evaluated (K Begum et al., Parasitol Res 2003 90-3, 221-4) for ten metalloporphyrins based on gallium, silver, tin, chromium, managanese, aluminium. In trophozoite lysate­mediated heme polymerization assays, SnPPIX, GaPPIX and GaDPIX exerted potent inhibitory activity similar to that of chloroquine. Children and adults living in areas with high Plasmodium falciparum endemicity have significantly elevated levels of immunoglobulin (P Perlman et al., Infection and Immunity, 1997 Jan, 116-121). Activated lymphocytes induce B cells to produce immunoglobulin. Several gallium(III) complexes have been tested at the Washington University against Plasmodium falciparum strains and some had IC(50) values of 80 nM in a CQ-sensitive strain (S Harpstrife et al., Inorganic Chemistry, 42-7, 2003, 2294-300). At the same university in 1997 already a Ga(III) complex had been developed, targeting chloroquine resistant organisms. The compound was shown to directly inhibit heme polymerization (V Sharma et al, Chem Commun 1997, 2223-2224). The interaction of gallium with artemisinin has not been studied. But several authors have described artemisinin-transferrin conjugates (I Nakase et al., Cancer letters, 2009 274, 290-8). Schisostoma worms are hematophagous parasites like Plasmodium. First trials in Africa on 50 persons show that after one week treatment with Artemisia annua capsules the worms have disappeared in the urine. Schisostomiasis (or bilharzia) kills 300 000 people per year. Whatever the mechanism of this herbal therapy, it is a tremendous hope for millions of Africans. Pierre Lutgen 20 Jan 2015