Plerixafor , New treatment approaches for patients suffering from frequent bacterial infections

Plerixafor

17 nov 2013

Scientists at A*STAR's Singapore Immunology Network (SIgN) have discovered the exact mode of action by plerixafor, a drug commonly prescribed to stimulate immune responses in patients suffering from neutropenia, which causes them to become prone to oral, skin, genital infections and in worst cases, a fatal whole-body infection. A better understanding of the drug's mechanism can improve its usage to more effectively reduce risk of infections in these patients.

Scientists at SIgN employed cutting-edge imaging techniques to analyze the effects of plerixafor on the white blood cell activity in the study which was published in the Journal of Experimental Medicine (JEM).

Neutrophil Mobilization via Plerixafor

Neutropenia is a condition characterized by the lack of a type of white blood cells, also known as neutrophils, in one's blood circulation. Plerixafor increases the concentration of these white blood cells in the blood by inhibiting a protein called CXCR4. This inhibition prevents neutrophils in the blood stream from returning to the bone marrow, which is the primary compartment where the white blood cells are stored and released. It is therefore commonly accepted that the efficacy of the drug arises only from the release of these white blood cells from the bone marrow.

However, scientists at SIgN found that the inhibition of CXCR4 by the drug actually plays a dual role - It increases the neutrophil count in the blood through their release from the lungs, while simultaneously promoting their retention in the blood stream. Discovery of this additional mode of action not only provides a deeper understanding on the drug's mechanism, it also contributes to a more effective utilization of the drug. The ground-breaking study creates the possibility of using a combined drug treatment to maximise release of white blood cells from both the bone marrow and the lungs. The approach may be more effective in reducing the risk of bacterial infections in neutropenic patients.

The team leader, Dr Ng Lai Guan from SIgN said, "We have identified the precise mechanisms of plerixafor treatment, which has important implications on its usage. We can understand through this study the effectiveness or limitations of the drug on certain patients and thereafter craft new clinical approaches to better treat them. Our study forms a conceptual framework to establish improved therapeutic strategies for neutropenia."

Acting Executive Director of SIgN, Associate Professor Laurent Rénia, said, "Basic research as such is important for us to fully understand how drugs work, so that we can put them to best use. This is a study which can potentially be translated into clinical applications to impact the health and lives of neutropenic patients."

Plerixafor (rINN and USAN, trade name Mozobil) is an immunostimulant used to mobilize hematopoietic stem cells in cancer patients. The stem cells are subsequently transplanted back to the patient. The drug was developed by AnorMED which was subsequently bought by Genzyme.

Three of the four nitrogen atoms of the macrocycle 1,4,8,11-tetraazacyclotetradecan are protected with tosyl groups. The product is treated with 1,4-dimethoxybenzene or 1,4-bis(brommethyl)benzene and potassium carbonate in acetonitrile. After cleaving of the tosyl groups with hydrobromic acid, plerixafor octahydrobromide is obtained.Bridger, G.; et al. (1993). "Linked cyclic polyamines with activity against HIV. WO/1993/012096".

The molecule 1,1′-[1,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane], consisting of two cyclam rings linked at the amine nitrogen atoms by a 1,4-xylyl spacer, was first synthesised by Fabbrizzi et al. in 1987 to carry out basic studies on the redox chemistry of dimetallic coordination compounds. Then, it was serendipitously discovered by De Clercq that such a molecule, could have a potential use in the treatment of HIV[2] because of its role in the blocking of CXCR4, a chemokine receptor which acts as a co-receptor for certain strains of HIV (along with the virus's main cellular receptor, CD4).Development of this indication was terminated because of lacking oral availability and cardiac disturbances. Further studies led to the new indication for cancer patients

Plerixafor has orphan drug status in the United States and European Union for the mobilization of hematopoietic stem cells. It was approved by the U.S. Food and Drug Administration for this indication on December 15, 2008. In Europe, the drug was approved after a positive Committee for Medicinal Products for Human Use assessment report on 29 May 2009. The drug was approved for use in Canada by Health Canada on December 8, 2011

BI 201302 a 15-membered macrocyclic hepatitis C virus (HCV) protease inhibitor

A highly convergent large scale synthesis of a 15-membered macrocyclic hepatitis C virus (HCV) protease inhibitor BI 201302 was achieved, in which the key features are the practical macrocyclization by Ru-catalyzed ring-closing metathesis (0.1 mol % Grela catalyst, 0.1–0.2 M concentration) and the efficient sulfone-mediated SNAr reaction.

Wei X * et al. Boehringer-Ingelheim Pharmaceuticals, Ridgeway, USA
A Highly Convergent and Efficient Synthesis of a Macrocyclic Hepatitis C Virus Protease Inhibitor BI 201302.

Org. Lett. 2013; 15: 1016-1019

http://pubs.acs.org/doi/abs/10.1021/ol303498m

Chemical Development, Boehringer-Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, Connecticut 06877, United States

Org. Lett., 2013, 15 (5), pp 1016–1019

DOI: 10.1021/ol303498m

Significance

The synthesis of the HCV protease inhibitor BI 201302 features an efficient ruthenium-catalyzed ring-closing metathesis reaction (0.1–0.2 M) requiring only 0.1 mol% of the Grela catalyst E to generate the 15-membered macrocycle F. This enhanced efficiency was achieved by installing a Boc substituent on the nitrogen of fragment D.

Comment

The SNAr reaction using a phenylsulfonyl leaving group in quinoline derivative H was more efficient than the reaction with the corresponding chloride (92% vs 40% yield). Potassium 3,7-dimethyl-3-octanoxide (KDMO) was used as a base instead of t-BuOK because transcarbamoylation byproducts (1–2%) were easily removed by crystallization.

Gossypol a male antifertility agent with antispermatogenic activity and has been shown to contain anitumor, anitviral, and antioxidant properties

Gossypol

2,2-Bis(1,6,7-trihydroxy-3-methyl-5-isopropyl-8-formylnaphthalene)

This ridiculously named molecule is found in cotton seeds. It was used as a male contraceptive in China, but was never used in the West (and may have since been banned in China as well), since its effects were permanent in about 20% of patients! Its name originated from being present in the flowers of the Indian cotton plant Gossypium herbaceum L. Apart from its contraceptive effects, gossypol has properties that might make it useful in treating a number of ailments, including cancer, HIV, malaria and some bacterial/viral illnessness. Related to this molecule are the equally strangely named gossypetin and gossypin.

Gossypol is a natural phenol derived from the cotton plant (genus Gossypium). Gossypol is a phenolic aldehyde that permeates cells and acts as an inhibitor for several dehydrogenase enzymes. It is a yellow pigment.

Among other things, it has been tested as a male oral contraceptive in China. In addition to its contraceptive properties, gossypol has also long been known to possess antimalarial properties. Other researchers are investigating the anticancer properties of gossypol.

Biological properties

It has proapoptotic properties, probably due to the regulation of the Bax and Bcl2. It also reversibly inhibits calcineurin and binds to calmodulin. It inhibits replication of the HIV-1 virus.^[1] It is an effective protein kinase C inhibitor.^[2] It also causes low potassium levels, and thus causes temporary paralysis.

Biosynthesis

Gossypol is a terpenoid aldehyde, which is formed metabolically through acetate via the isoprenoid pathway.^[3] Sesquiterpene dimer undergoes a radical coupling reaction to form gossypol.^[4] Geranyl pyrophosphate (GPP) and IPP make sesquiterpene precursor, farnesyl diphosphate (FPP), for gossypol. The biosynthesis of gossypol is summarized in figure below. The biosynthesis begins with the 0 compound derived from GPP and IPP. Cadinyl cation (1) is oxidized to 2 by (+)-d-cadinene synthase. The (+)-d-cadinene (2) is involved in making the basic aromatic sesquiterpene unit, homigossypol, by oxidation, which generates the 3 (8-hydroxy-d-cadinene) with the help of (+)-d-cadinene 8-hyroxylase. At 5, the 3 goes through various oxidative processes to make 4 (deoxyhemigossypol), which is oxidized by one electron into hemigossypol (5,6,7) and then undergoes a phenolic oxidative coupling, ortho to the phenol groups, to form 8 (gossypol).^[5] The coupling is catalyzed by a hydrogen peroxide-dependent peroxidise enzyme, which results in the final product.^[5]

Contraceptive use

A 1929 investigation in Jiangxi showed correlation between low fertility in males and use of crude cottonseed oil for cooking. The compound causing the contraceptive effect was determined to be gossypol.

In the 1970s, the Chinese government began researching the use of gossypol as a contraceptive. Their studies involved over 10,000 subjects, and continued for over a decade. They concluded gossypol provided reliable contraception, could be taken orally as a tablet, and did not upset men's balance of hormones.

However, gossypol also had serious flaws. The studies also discovered an abnormally high rate of hypokalemia among subjects.^[6] Hypokalemia — low blood potassium levels — causes symptoms of fatigue, muscle weakness, and at its most extreme, paralysis. In addition, about 7% of subjects^{[citation needed]} reported effects on their digestive systems, and about 12%^{[citation needed]} had increased fatigue. Most subjects recovered after stopping treatment and taking potassium supplements. The same study showed taking potassium supplements during gossypol treatment did not prevent hypokalemia in primates.^[7] The potassium deficiency may also be a result of the Chinese diet or genetic predisposition. ^[8]

In the mid-1990s, the Brazilian pharmaceutical company Hebron announced plans to market a low-dose gossypol pill called Nofertil, but the pill never came to market. Its release was indefinitely postponed due to unacceptably high rates of permanent infertility^[]. 5-25% of the men remained azoospermic up to a year after stopping treatment.^[9] The longer the men had taken the drug and the higher their overall dosages, the more likely^{[citation needed]} they were to have lowered fertility or to become completely infertile.

Researchers have suggested gossypol might make a good noninvasive alternative to surgical vasectomy.^[10]

In 1986, the Chinese stopped research because of these side effects.

In 1998, the World Health Organization's Research Group on Methods for the Regulation of Male Fertility recommended the research should be abandoned. In addition to the other side effects, the WHO researchers were concerned about gossypol's toxicity: the toxic dose in primates is less than 10 times the contraceptive dose.^[11]This report effectively ended further studies of gossypol as a temporary contraceptive, but research into using it as an alternative to vasectomy continues in Austria, Brazil,Chile, China, the Dominican Republic, and Nigeria.

First identified as an anti-fertility agent in China in the 1950s, gossypol is also a component of cottonseed oil, which is used for cooking. It's derived from the stems, roots, and seeds of plants from the Malvaceae family. The cotton plant (Gossypium species) is the most common source. Gossypol is the active ingredient found in seeds and other parts of the plant; however, content varies significantly from specjes to species.

Gossypol exerts antifertility action by inhibiting sperm production and motility. It possesses antitumorigenic activity and may also have anti-human immunodeficiency virus properties. It's available as liquid extracts and tinctures.

Reported uses

Gossypol is used in China as a male contraceptive. It's also used topically as a spermicide.

Administration

Dosage is 20 mg by mouth every day for 2 to 3 months until the sperm count is decreased to less than 4 million/ml. The dosage is reduced to a maintenance ranging from 50 mg weekly to 75 to 100 mg twice a month.

Hazards

Adverse effects associated with gossypol include paralysis, circulatory problems, diarrhea, malnutrition, hypokalemia, muscle fatigue, muscle weakness, and hair discoloration.

Pregnant and breast-feeding patients shouldn't use this herb. Patients with renal insufficiency should use with caution.

Safety Risk Gossypol has been associated with heart failure and nephrotoxicity. There's increased risk of nephrotoxicity when gossypol is given with nephrotoxic drugs. Administration with potassium-wasting diuretics could lead to hypokalemia.

Clinical considerations

The contraceptive effect of gossypol in men is higher than 99%. Fertility usually returns to normal within 3 months of discontinuation; however, inhibition of spermatogenesis may persist in up to 20% of men 2 years after discontinuation.

Monitor serum electrolyte levels, especially potassium, creatinine, and BUN levels.

Monitor patient for muscle weakness and fatigue.

If using formulation containing alcohol, avoid using in patients taking disulfiram, metronidazole, cephalosporins, or any CNS depressants.

If patient is pregnant or breast-feeding, advise her not to use gossypol.

Inform men of the potential for permanent sterility after using oral gossypol.

Food and animal agricultural industries must manage cotton-derivative product levels to avoid toxicity. For example, only ruminant microflora can digest gossypol, but only to a certain level, and cottonseed oil must be refined.

A research team at Texas A&M University has genetically engineered cotton plants that contain very little gossypol in the seed, but still contain the compound in the stems and leaves. This provides protection against pests and diseases, while allowing the seed to be used for oil and meal for human consumption. The plants are modified by RNA interference, shutting down the genes for gossypol production in the seed, while leaving them unaffected in the rest of the plant. The resulting gossypol-free cottonseed is then suitable as a high-quality protein source suitable for consumption not only by cattle, but also by humans. Protein makes up 23% of the cottonseed. ^[12][13]

Gossypol is toxic to erythrocytes in vitro by stimulating cell death contributing to the side effect of hemolytic anemia.^[14]

References[edit]

1. Jump up^ Polsky, B; Segal, SJ; Baron, PA; Gold, JW; Ueno, H; Armstrong, D (1989). "Inactivation of human immunodeficiency virus in vitro by gossypol". Contraception 39 (6): 579–87.doi:10.1016/0010-7824(89)90034-6. PMID 2473865.
2. Jump up^ Gossypol (Gossipol). Bioscreening.net (last updated 2008-07-09). Retrieved on 2012-06-09.
3. Jump up^ Burgos, M.; Ito, S.; Segal, J. S.; Tran, T. P. (1997). "Effect of Gossypol on Ultrastructure of Spisula Sperm". Biol. Bull. 193: 228–229.
4. Jump up^ Heinstein, P. F. ; Herman, L. D.; Tove, B. S.; Smith, H. F. (1970). "Biosynthesis of Gossypol". J. Biol. Chem. 245 (18): 4658–4665. PMID 4318479.
5. ^ Jump up to:^a b Dewick, P. M. Medicinal Natural Product: A Biosynthetic approach. 3rd ed., 2008 ISBN 0-470-74167-8
6. Jump up^ Gossypol. Malecontraceptives.org (2011-07-27). Retrieved on 2013-03-30.
7. Jump up^ Gossypol. Malecontraceptives.org (2011-07-27). Retrieved on 2013-03-30.
8. Jump up^ Gossypol. Malecontraceptives.org (2011-07-27). Retrieved on 2013-03-30.
9. Jump up^ Gossypol. Malecontraceptives.org (2011-07-27). Retrieved on 2013-03-30.
10. Jump up^ Coutinho, F. M. (2002). "Gossypol: a contraceptive for men". Contraception 65 (4): 259–263. doi:10.1016/S0010-7824(02)00294-9. PMID 12020773.
11. Jump up^ Gossypol. Malecontraceptives.org (2011-07-27). Retrieved on 2013-03-30.
12. Jump up^ Cottonseed Protein: From Farmers to Your Family Table. Medgadget.com (2006-11-22). Retrieved on 2012-06-09.
13. Jump up^ Walsh, Brian. Hungry? How About Some Protein-Rich Cotton..., Time Magazine, September 14, 2009, p. 54
14. Jump up^ Zbidah, M; Lupescu, A; Shaik, N; Lang, F (2012). "Gossypol-induced suicidal erythrocyte death". Toxicology 302 (2–3): 101. doi:10.1016/j.tox.2012.09.010. PMID 23041711.

• Ibragimov, B. T.; Talipov, S. A.; Aripov, T. F.; Sadykov, A. S. (1990). "Inclusion complexes of the natural product gossypol. Crystal structure of the 2:1 complex of gossypol withm-Xylene". Journal of Inclusion Phenomena and Molecular Recognition in Chemistry 8 (3): 323. doi:10.1007/BF01041888.

Biosynthesis of gossypol from hemigossypol (Stipanovic et. al. 2005)

the compound is derived from hemigossypol through a free radical coupling reaction (Stipanovic et. al. 2005). This coupling reaction produces the two enantiomers (+)-gossypol and (-)-gossypol, depending on the orientation around the binaphthyl bond.

The toxicity of gossypol has made it an area of interest in plant research. When ingested orally, its toxicity can lead to anorexia, severe weight loss, and even death (Eagle 1949). The (-)-gossypol enantiomer will enter the cell and act as an inhibitor of dehydrogenase enzymes involved electron transport (Benz et. al. 1990), which ultimately leads to necrosis of the tissue.

There are also advantageous properties of gossypol to the cotton plant, especially in medicinal uses. (-)-gossypol is a potential aid in cancer research due to its inhibition of cell growth (Band et. al. 1989; Blackstaffe et. all 1997; Shelley et. al. 1999). This is accomplished by reduction of ATP by the uncoupling of oxidative phosphorylation in electron transport (Benz et. al. 1990). HIV research has shown that (-)-gossypol shows anti-HIV-1 activity (Lin et. al. 1993).This enantiomer can be used as an antiamoebic agent (Gonzales-Graza et. al. 1992). There have also been studies in China to suggest its use as a male contraceptive (Matlin et. al. 1985; Lindberg et. al. 1987; Wang et. al. 1987; Yao et. al. 1987).

Research shows that (+)-gossypol is useful to cotton as a natural protection against insects and pathogens. For example, Helicoverpa armigera larvae mature slowly and have a lower survival rate on cotton plants with (+)-gossypol (Yang et. al. 1999). It can also be consumed by nonruminant animals without expressing any signs of toxicity (Stipanovic et. al. 2005).

Cotton is well known as the leading fiber crop in the world (Lusas and Jividen 1987), but it's potential as a food crop has often been overlooked (Alford et. al. 1996). It has actually been classified as the second best potential source for plant proteins and the fifth best oil-producing plant (Texier 1993). Gossypol's toxicity restrains the potential of using cotton as a food crop, which is why emphasis has been placed on breeding for cotton with reduced amounts of gossypol in its seed (Vroh et. al. 1999). With low gossypol in the seed it can be safely used to produce cotton oil.

Zerp et al. Radiation Oncology 2009 4:47   doi:10.1186/1748-717X-4-47

Synonym: AT 101; 1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'-binaphthyl-8,8'-dicarbaldehyde

Application: An inhibitor of PKC, Bcl-2, 5-LO, and 12-LO

CAS Number: 303-45-7

Purity: ≥90%

Molecular Weight: 518.56

Molecular Formula: C30H30O8

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Description

Gossypol is a male antifertility agent with antispermatogenic activity and has been shown to contain anitumor, anitviral, and antioxidant properties. Gossypol is a reversible inhibitor of PP2B (protein phosphatase 2B), mitotic kinesin Eg5, and PKC (protein kinase C). Gossypol also potentially inhibits PAF-R, Bcl-2, 5-LO (5-lipoxygenase), 12-LO, and leukotriene-induced guinea pig parenchyma contractions. Gossypol is an inhibitor of Bcl-xl, PKD and p107.

Technical Information

Appearance: Off-white to yellow crystalline solid

Physical State: Solid

Derived from: Gossypium genus, Malvaceae

Solubility: Soluble in 100%ethanol (25 mg/ml), DMF (25 mg/ml), acetone, DMSO (25 mM), methanol (2 mg/ml), ether, chloroform, sodium carbonate, and dilute aqueous solutions of ammonia . Insoluble in water.

Storage: Store at -20° C

Melting Point: 191-193 ºC

Boiling Point: 707.89 °C at 760 mmHg

Density: 1.40 g/cm3

Refractive Index: n20D 1.74

IC50: mitotic kinesin Eg5: IC50 = 10.8 µM; RBL-1, 5-lipoxygenase: IC50 = 0.3 µM; 12-lipoxygenase: IC50 = 0.7 µM; PC-3 (Prostate carcinoma cells) : EC5050 = 17.8 µM (Homo sapiens ); PC-3 (Prostate carcinoma cells) : EC5050 = 3.3 µM (Homo sapiens ); Transitional endoplasmic reticulum ATPase : IC50 = 5.39 µM (Homo sapiens ); Induced myeloid leukemia cell differentiation protein Mcl-1 : IC50 = 1.36 µM (Homo sapiens ); Estrogen receptor beta : IC50 >50 µM (Homo sapiens ); Apoptosis regulator Bcl-2 : IC50 = 0.5 µM (Homo sapiens ); HCT-116 (Colon carcinoma cells) : IC50 = 3.3 µM (Homo sapiens ); Malate dehydrogenase mitochondrial Malate dehydrogenase mitochondrial : IC50 = 2.8 µM (Sus scrofa ); MDA-MB-231 (Breast adenocarcinoma cells) : IC50 = 1.9 µM (Homo sapiens ); Lactate dehydrogenase : IC50 = 2.64 µM (Plasmodium falciparum ); Malate dehydrogenase : IC50 = 2.03 µM (Plasmodium falciparum )

Ki Data: Induced myeloid leukemia cell differentiation protein Mcl-1 : Ki= 0.18 µM (Homo sapiens ); Apoptosis regulator Bcl-X : Ki= 0.48 µM (Homo sapiens ); Apoptosis regulator Bcl-2 : Ki= 0.32 µM (Homo sapiens ); Aldose reductase : Ki= 0.5 µM (Homo sapiens ); Apoptosis regulator Bcl-2 : Ki= 0.17 µM (Homo sapiens ); Apoptosis regulator Bcl-W : Ki= 17.7 µM (Homo sapiens )

3,4-Dimethoxy-2-isopropylbenzaldehyde (I) could be condensed with diethyl succinate (II) to produce 2-(2-isopropyl-3,4-dimethoxybenzylidene)succinic acid monoethyl ester (III) in the presence of NaH in refluxing benzene, and the yielding product is cyclized by treatment with acetic anhydride and sodium acetate in refluxing acetic acid and hydrolyzed with NaOH in refluxing methanol affording acid (IV). Next, (IV) is reduced with LiAlH₄ in ether to give 3-hydroxymethyl-5-isopropyl-6,7-dimethoxy-1-naphthol (V), and the compound produced is hydrogenated with H₂ over Pd/C in methanol with some HCl affording 3-methyl-5-isopropyl-6,7-dimethoxy-1-naphthol (VI). Thermal dimerization of (VI) in the condition of heating at 215 C yields 2,2-bis(3-methyl-5-isopropyl-6,7-dimethoxy-1-naphthol) (VII).