American Journal of Therapeutics 8, 321–328 (2001)
`
`Boron Therapeutics on the Horizon
`
`Michael P. Groziak
`
`No pharmaceutical based on boron has yet made it to market, but this may soon change. The new
`millennium has brought with it some unique classes of bioactive boron compounds that are suffi-
`ciently mature in development to be considered significant and timely advances in their respective
`chemotherapeutic areas. Because boron is seldom seen as a constituent of a bioactive agent, this
`review relates some of the pertinent biologic and physiologic properties of boron and then describes
`in detail those boron-based agents clearly visible on the therapeutic horizon. Highlighted agents
`include boronic acids and boron heterocycles as potent proteasome inhibitors, ␤-lactamase inhibi-
`tors, dipeptidyl peptidase inhibitors, inositol trisphosphate receptor modulators, antibacterials, and
`antiestrogens. As these new agents are welcomed into the therapeutic armamentarium, others will
`surely follow and the prescribing clinician will already have an awareness and appreciation of the
`unique benefits that these compounds have to offer.
`
`Keywords: boron, drug development, proteasome, ␤-lactamase, anticoagulant, antithrombotic, di-
`peptidyl peptidase IV, inositol trisphosphate receptor, antibacterial, antiestrogen.
`
`INTRODUCTION
`
`Boron, the element immediately to the left of carbon in
`the periodic table, has some unique and potentially
`valuable properties to offer to medicine, but unfortu-
`nately it has been greatly underutilized in therapeutic
`agent development to date. There are two principal
`reasons for this. The first is that very few boron-
`containing natural products are available to serve as
`an intellectual spark for medicinal chemists in their
`drug-design efforts, and to make matters worse, these
`turn out to be rather poor models. The ionophoric
`macrodiolide antibiotics boromycin,1,2 aplasmomy-
`cin,3–5 and tartrolon B6–8 are all carbon/oxygen-based
`macrocycles that tightly yet reversibly complex boric
`acid in its borate conjugate base form through a net-
`work of four dissociable B-O bonds. These tetrahedral
`borate anion complexes are such potent potassium ion
`carriers that they are highly toxic to both bacteria and
`
`Pharmaceutical Discovery Division, SRI International, Menlo
`Park, CA.
`Supported by NIH grant NIGMS GM44819 and US Army grant
`USAMRAA DAMD17–00–1-0661.
`Address for correspondence: Pharmaceutical Discovery Division,
`SRI International, Menlo Park, CA 94025–3493, USA; e-mail:
`michael.groziak@sri.com
`
`1075–2765 © 2001 Lippincott Williams & Wilkins, Inc.
`
`mammalian cells. They are the only boron-containing
`natural products known. No boronic acid natural
`product has ever been found, and it may be that na-
`ture simply lacks the biosynthetic enzyme machinery
`needed to form a carbon-boron bond. This, in turn,
`suggests that nature might also lack the metabolic en-
`zymes needed to break them. If this turns out to be
`true, it would be highly significant to the development
`of boronic acid-based therapeutic agents.
`The second reason for the underutilization of boron
`in drug development is that very few organic chemists
`have explored the construction of stable boron-
`containing molecular platforms on which new bioac-
`tive agents could be built.9 The deliberate engineering
`of boron in a hydrolysis-resistant and charge-useful
`manner into such a platform requires considerable
`thought and planning in molecular design. For ex-
`ample, whereas anionic tetrahedral borates like those
`in the macrodiolide natural products are of very lim-
`ited utility as molecular design fragments because of
`their charge, the carbon-substituted forms of boric
`acid B(OH)3 are considerably more useful—but pri-
`marily just in monosubstituted form. This form is typi-
`fied by the very weakly acidic boronic acids RB(OH)2,
`which are water stable and neutral compounds at
`physiologic pH and thus are quite well suited for
`pharmaceutical agent design. Disubstitution, con-
`versely, gives the borinic acids R2BOH, which are
`
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`

`322
`
`GROZIAK
`
`acidic compounds existing largely as tetrahedral an-
`ionic borinates at physiologic pH. Trisubstitution
`gives the triorganoboranes R3B, which are extremely
`acidic and for all practical purposes useless in drug
`design. Thus, it is not at all surprising to find that most
`of the boron-based therapeutics currently on the hori-
`zon are either boronic acids themselves or boron het-
`erocycles that are simply internally complexed ver-
`sions of boronic acids.
`
`BIOLOGICAL ASPECTS OF BORON
`
`Boronic acids are fairly common and easily prepared
`synthetic organic compounds. Many are commercially
`available, and none to date has been found to be un-
`usually toxic. They are stable under physiologic con-
`ditions but can be induced under laboratory condi-
`tions to undergo chemical deboronation via either
`hydrolysis or oxidation. The former generates a hy-
`drocarbon and the latter an alcohol from the organic
`fragment, but both produce boric acid. It follows, then,
`that no matter whether a boronic acid–based thera-
`peutic agent is actively metabolized or simply under-
`goes chemical degradation in vivo, the production of
`boric acid is not to be unanticipated. It is therefore
`important to be aware of some of its biologic and
`physiologic properties.
`Boron, present solely as boric acid or its borate salts
`in nature, is a micronutrient soil component essential
`for growth in vascular plants,10,11 which take it up by
`the roots.12 It is required by diatoms, but not fungi or
`bacteria, and it has been shown to stimulate growth in
`yeast.13 A specific biochemical function for boron in
`mammals has yet to be determined, but dietary
`supplementation with it is known to increase signifi-
`cantly plasma steroid hormones,14 alter plasma lipid
`metabolites, and improve bone strength,15 thus having
`implications in clinical conditions like arthritis.16 Bo-
`ron nutriture may be important for brain and psycho-
`logical function,17 and indeed it may be important
`throughout the life cycle.18 A recent study indicates
`that boron metabolism in humans may be subject to
`genetic regulation.19 Evidence for its role as an essen-
`tial nutrient for humans steadily accumulated during
`the past decade,20–22 and it now seems likely that di-
`etary boron merits some form of guidance under a
`recommended dietary allowance.23–25 Earlier this
`year, the US Food and Nutrition Board set a Tolerable
`Upper Intake Level (UL) for boron at 20 mg per
`day.25A In the United States, coffee and milk account
`for the largest total boron intake because of their high
`consumption rate, but wine, raisins, and peanuts and
`other nuts actually have a higher boron content.26 In-
`terestingly, boron in drinking water was once thought
`
`American Journal of Therapeutics (2001) 8(5)
`
`to play a beneficial role in the pathology of dental
`caries,27 it has attracted some attention in alternative
`medicine for treating osteoarthritis,28 and it has been
`used by athletes as a nutritional supplement for build-
`ing muscle mass.29
`Boric acid (LD50 of 3450 mg/kg orally in the mouse
`and 2660 mg/kg orally in the rat) and its simple borate
`salts like borax have been studied in great detail and
`pose no significant toxicity threat,30 even though there
`is considerable exposure from consumer products.31
`Serious poisoning of humans with boric acid is un-
`likely to result from a single acute ingestion,32 and
`aggressive treatment is not necessary33 unless there
`has been inadequate urine flow and dehydration for
`several days.34 Boric acid does not associate strongly
`with serum proteins but instead rapidly diffuses to the
`extravascular space without accumulating in tissues
`and is excreted efficiently via glomerular filtration.35
`Pregnancy has been shown to have little or no effect
`on the renal clearance of boric acid in both rats36 and
`humans.37 Additional health-related information on
`boric acid and simple organoboron compounds can
`be found in the contributions to the International
`Symposia on the Health Effects of Boron and Its
`Compounds38,39 and in specialized reviews of the
`chemistry and biology of simple inorganic and organic
`boron compounds.40,41
`
`PROTEASOME INHIBITORS
`
`Boronic acids interconvert with ease between a neutral
`trigonal planar form and an anionic tetrahedral borate
`one, and this property enables them to potently inhibit
`biochemical acyl group transfer reactions, especially
`hydrolyses like those catalyzed by the enzymes chy-
`motrypsin, trypsin, thrombin, and other proteases. Bo-
`ronic acid–based protease inhibition first emerged
`three decades ago with the discovery of good inhibi-
`tors of chymotrypsin.42–45 A few of the boronic acids
`effect simple competitive inhibition from their anionic
`tetrahedral borate form, but most undergo a reversible
`yet strong covalent attachment from their neutral tri-
`gonal planar form to a protease active site nucleo-
`phile—usually a serine residue.
`The proteasome is the major nonlysosomal endo-
`protease in cells, where it generates antigenic peptide
`ligands for the major histocompatibility complex
`(MHC) class I proteins. Suppressing the production of
`antigens for cytotoxic T cells by inhibiting the protea-
`some, therefore, is an important approach to modify-
`ing the cytotoxic immune response. This has obvious
`applications in the areas of transplant rejection and
`autoimmune disease.46 Inhibiting the proteasome has
`applications in cancer chemotherapy as well. For ex-
`
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`BORON THERAPEUTICS ON THE HORIZON
`
`323
`
`ample, the dipeptide boronic acids like PS-341 (Fig.
`1A) represent a new class of proteasome inhibitor47
`that are entering early-phase clinical trials as antican-
`cer drugs.48–50 PS-341, an N-pyrazinylcarbonylated
`derivative of the dipeptide boronic acid Phe-Leu-
`B(OH)2, is potently cytotoxic against cultured MCF-7
`human breast cancer cells with an IC90 of 50 nmol/L
`on 24 hours of exposure to the drug. The combination
`of proteasome inhibition with conventional chemo-
`therapy may have significant potential in overcoming
`the high incidence of chemotherapy resistance.51 In
`addition, the ability to suppresses ␤-amyloid peptide
`(A beta) secretion from cultured cells has been found
`to correlate extremely well with a peptide boronic ac-
`id’s potency at inhibiting the proteasome. New boron-
`based chemotherapeutics for Alzheimer’s disease may
`therefore be forthcoming.52
`
`ANTICOAGULANTS
`
`In the anticoagulant field, the clinically used heparins
`and the vitamin K antagonist warfarin may soon be
`joined by new boronic acid inhibitors of the trypsin-
`like protease thrombin and the coagulation factor Xa.
`Several of these types of compounds are now moving
`into clinical trials.53 The inhibition of thrombin by bo-
`ronic acids has been under development for nearly a
`decade,54–59 with the aim to produce a drug to comple-
`ment hirudin. The development of orally bioavailable
`boronic acid inhibitors of coagulation factor Xa is
`more recent,60 and interest is high because cell signal-
`ing by Xa contributes to pro-inflammatory responses
`in vivo.
`
`␤-LACTAMASE INHIBITORS
`
`Bacterial resistance to the ␤-lactam antibiotics has cre-
`ated a pressing need for new therapeutic agents for
`the treatment of ␤-lactam–resistant infections. Many
`␤-lactamases depend on an essential active-site serine
`residue to effect catalysis, making them ideal
`targets for potent inhibition by boronic acids. Early
`on, simple boronic acids and even boric acid itself
`were found to competitively inhibit a ␤-lactamase
`from Bacillus cereus.61 Soon afterward, boronic acids
`active against ␤-lactamases from Pseudomonas aerugi-
`nosa and Escherichia coli62 and from Citrobacter diversus
`and P. aeruginosa63 were uncovered. Using a rational
`design approach, the simple boronic acids were struc-
`turally elaborated to resemble the drugs penicillin G
`and methicillin, and these new compounds were
`found to potently inhibit ␤-lactamases from B. cereus
`and P. aeruginosa.64
`An x-ray crystal structure of 3-aminophenylboronic
`acid bound by E. coli AmpC ␤-lactamase was ob-
`tained, and this was used as a guide to screen a large
`number of other boronic acids for activity.65 One of the
`most potent uncovered in this manner was the benzo-
`thiophene boronic acid known as BZBTH2 (Fig. 1B),
`inhibitory with a Ki of 27 nmol/L against this intrin-
`sically resistant ␤-lactamase.66 In a follow-up study,
`the x-ray crystal structure of BZBTH2, itself bound by
`AmpC ␤-lactamase, was solved to assist in the struc-
`ture-based development of third-generation boronic
`acid inhibitors. At the same time, BZBTH2 properties
`that are rather un-␤-lactam in nature were discovered,
`namely, that this agent is unaffected by two common
`
`Fig. 1. Chemical structures of the proteasome inhibitor PS-341 (A), the ␤-lactamase inhibitor BZBTH2 (B), the ␤-lacta-
`mase inhibitor (1R)-1-benzamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (C), the dipeptidyl peptidase IV in-
`hibitor ProboroPro (D), the inositol 1,4,5-trisphosphate receptor modulator 2-APB (E), the enoyl acyl carrier protein
`reductase inhibitor benzodiazaborine (F), and a boron estrogen mimic (G).
`
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`324
`
`GROZIAK
`
`resistance mechanisms, and, furthermore, it does not
`induce the expression of AmpC.67
`One of the first rationally designed boronic acid in-
`hibitors of the TEM-1 ␤-lactamase from E. coli was a
`very potent compound, with a Ki of 110 nmol/L.68 An
`x-ray crystal structure of the bound inhibitor was ob-
`tained, and even more potent compounds were de-
`signed based on that structure.69 This effort produced
`(1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethyl bo-
`ronic acid (Fig. 1C), a compound closely resembling
`the best known TEM-1 ␤-lactamase substrate, benzyl-
`penicillin (penicillin G).70 This boronic acid is inhibi-
`tory with a Ki of 5.9 nmol/L, an amazing potency
`supporting the widespread contention that boronic ac-
`ids are superior to all other kinds of inhibitor species,
`as was demonstrated for the class C ␤-lactamase of
`Enterobacter cloacae P99.71
`
`DIPEPTIDYL PEPTIDASE IV
`INHIBITORS
`
`The serine protease dipeptidyl peptidase IV (DP-IV,
`also known as CD26 and Tp103) is a 103-kd activating
`molecule expressed on the surface of human T lym-
`phocytes. It is inhibited quite effectively by a boron
`compound known as ProboroPro, the dipeptide bo-
`ronic acid (D)Pro-(D)Pro-B(OH)2 (Fig. 1D).72–76 In con-
`trast to other serine proteases, DP-IV hydrolyzes the
`normally inhibitory N-peptidyl-O-acyl hydroxyl-
`amines, thus hinting at a catalytic mechanism for this
`proline-specific enzyme that is somehow different
`from the others. Proline-containing peptide boronic
`acids are usually somewhat unstable, but the acyl pyr-
`rolidides like ProboroPro have a good stability and
`typically inhibit DP-IV in the micromolar and some-
`times even nanomolar range. DP-IV is virtually absent
`on resting T cells, but after activation, it is strongly
`expressed and becomes involved in signal transduc-
`tion during the immune response. Some of the newly
`developed boronic acid DP-IV inhibitors potently sup-
`press T-cell proliferation.
`
`INS(1,4,5)P3 RECEPTOR
`MODULATORS
`
`in hepatocytes, but curiously by a mechanism that
`does not involve the Ins(1,4,5)P3 receptor.81 2-APB is an
`unusually stable borinic acid compound because of
`the ethanolamine side chain. This side chain intramo-
`lecularly complexes to the acidic boron center and
`forms a five-membered ring zwitterion (see also Fig.
`1E), which is quite stable. It is also nonpolar because,
`with no net charge, it is electrically neutral. Together
`with the presence of the two lipophilic phenyl groups,
`this explains why this molecule so readily penetrates
`membranes. At present, 2-APB is a valuable pharma-
`cologic tool in sleep research since the Ins(1,4,5)P3-
`induced release of calcium appears to play a role in the
`resetting of the mammalian circadian clock in the su-
`prachiasmatic nucleus.82 Clearly, the class of 2-APB-
`like compounds has the potential of producing valu-
`able therapeutics for the treatment of sleep disorders.
`
`ENOYL ACYL CARRIER PROTEIN
`REDUCTASE INHIBITORS
`
`For more than two decades, it has been known that
`benzodiazaborine (Fig. 1F) and other ring-fused sulfo-
`nylated diazaborine heterocycles possess antibacterial
`properties, particularly against gram-negative organ-
`isms.83 These endocyclic boron-containing com-
`pounds are simply aromatic boronic acids in which
`the boron is additionally held clamped by a covalent
`bond (the B-N) to a side chain. Early indications were
`that these compounds affected lipopolysaccharide bio-
`synthesis,84–92 and recent structural studies have es-
`tablished that the biomacromolecular target is in fact
`enoyl acyl carrier protein reductase (ENR), the
`NAD(P)H-dependent enzyme responsible for cata-
`lyzing a late step on the fatty acid biosynthetic path-
`way.93–97 Diazaborine inhibitors of ENR form a cova-
`lent B-O bond with the 2⬘-hydroxyl group of the co-
`factor NAD’s ribose unit, thus assembling a tightly yet
`noncovalently bound borate-based bisubstrate analog
`species at the active site. Interestingly, ENR is the very
`same biomacromolecular target of the commonly used
`broad-spectrum (bacteria, fungi, viruses) bacteriostat-
`ic germicide triclosan98–101 and even, oddly enough,
`the well-known antituberculosis drug isoniazid.
`
`ESTROGEN MIMICS
`
`The simple borinic acid 2-aminoethyl diphenyl bori-
`nate (2-APB, Fig. 1E) was discovered to be a novel
`membrane-permeable modulator of the inositol
`1,4,5-trisphosphate receptor.77 2-APB inhibits the
`Ins(1,4,5)P3-induced release of calcium78–80 and appears
`to inhibit the store operated calcium channels (SOCs)
`
`Our own laboratory has investigated the chemical and
`structural properties of a variety of benzodiazaborines
`formed via intramolecular dehydration in 2-acyl-
`amino- and amidino-substituted benzeneboronic ac-
`ids102 and in oximes and hydrazones of 2-formylben-
`zeneboronic acid.103,104 By examining the structural
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`BORON THERAPEUTICS ON THE HORIZON
`
`325
`
`features of the latter compounds, we recognized the
`opportunity to design new variants of these boron het-
`erocycles that would be ultra-high fidelity estrogen
`structural mimics. In particular, we saw that careful
`molecular engineering could produce very close struc-
`tural mimics of A-ring aromatic estrogens like estra-
`diol and estrone and AB-ring aromatic equilenins like
`some of the components of Premarin, the conjugated
`equine estrogens used extensively for estrogen re-
`placement therapy and prevention of osteoporosis and
`cardiovascular disease in postmenopausal women.
`Our first prototype (Fig. 1G) was a boron heterocycle
`mimic of estradiol or dihydroequilenin O17-methyl
`ether featuring a strong intramolecular hydrogen
`bond that assembles a “virtual,” flexible steroid-like
`C-ring. An x-ray crystal structure confirmed the re-
`markable estrogen-like shape adopted by this com-
`pound.105 This and some of the other early prototypes
`that had a biorecognition-critical estrogen A-ring phe-
`nolic hydroxyl group were examined for their antipro-
`liferative activity against cultured MCF-7 human
`breast cancer cells. The IC50 values near 5 ␮mol/L
`found for all these compounds provides a strong im-
`petus for our current ongoing efforts to develop these
`unique boron-based compounds into therapeutics for
`treating breast cancer, likely as antiestrogens.
`
`OTHERS
`
`Other boron-based compounds exhibit intriguing in
`vitro activity and thus show some promise of evolving
`into useful therapeutics in the future. Examples in-
`clude variants of benzodiazaborines found active
`against Mycobacterium tuberculosis H37Rv,106 acyclic
`nucleoside boronic acid derivatives targeted at HIV,107
`tetrapeptide boronic acids found to inhibit HIV-1 pro-
`tease,108 benzothiazoline boron complexes with anti-
`microbial properties,109 and 3-aminophenylboronic
`acid (APBA), found to inhibit both the Streptomyces
`griseus NAD+-glycohydrolase and ADP-ribosyltrans-
`ferase enzymes.110 The ongoing development of these
`bioactive agents will be well worth monitoring along
`side those that are further along in their development
`as useful therapeutics.
`
`CONCLUSION
`
`The novel classes of boron-based compounds de-
`scribed above are rapidly maturing into potent thera-
`peutics in their own right, and prescribing clinicians
`need to be aware that they are coming on line. The fact
`that they are based on the unusual element boron is no
`
`cause for concern because it is only toward the end of
`the past millennium that organic chemists have
`learned to construct useful platforms with it and me-
`dicinal chemists have learned to appreciate its value.
`
`REFERENCES
`
`1. Hütter R, Keller-Schierlein W, Knüsel F, et al: Stoff-
`wechselprodukte von Mikroorganismen. Boromycin.
`Helv Chim Acta 1967;50:1533–1539.
`2. Kohno J, Kawahata T, Otake T, et al: Boromycin, an
`anti-HIV antibiotic. Biosci Biotechnol Biochem 1996;60:
`1036–1037.
`3. Okami Y, Okazaki T, Umezawa H: Studies on marine
`microorganisms. V. A new antibiotic, aplasmomycin,
`produced by a streptomycete isolated from shallow sea
`mud. J Antibiot (Tokyo) 1976;29:1019–1025.
`4. Nakamura H, Iitaka Y, Kitahara T, et al: Structure of
`aplasmomycin. J Antibiot (Tokyo) 1977;30:714–719.
`5. Chen TS, Chang CJ, Floss HG: Biosynthesis of the bo-
`ron-containing antibiotic aplasmomycin. Nuclear mag-
`netic resonance analysis of aplasmomycin and desboro-
`aplasmomycin. J Antibiot (Tokyo) 1980;33:1316–1322.
`6. Schummer D, Irschik H, Reichenbach H, et al: Antibi-
`otics from gliding bacteria, LVII. Tartrolons: new bo-
`ron-containing macrodiolides from Sorangium cellu-
`losum. Liebigs Ann Chem 1994;3:283–289.
`7. Irschik H, Schummer D, Gerth K, et al: The tartrolons,
`new boron-containing antibiotics from a myxobacte-
`rium, Sorangium cellulosum. J Antibiot (Tokyo) 1995;48:
`26–30.
`8. Berger M, Mulzer J: Total synthesis of tartrolon B. J Am
`Chem Soc 1999;121:8393–8394.
`9. Groziak MP: Boron heterocycles as platforms for build-
`ing new bioactive agents. In: Progress in Heterocyclic
`Chemistry. (Eds. Gribble GW, Gilchrist TL). Elsevier Sci-
`ence, Oxford, 2000, pp1–21.
`10. Marschner H: Function of mineral nutrients: micronu-
`trients. 9.7 Boron. In: The Mineral Nutrition of Higher
`Plants, 2nd ed. Academic Press, San Diego, 1995,
`pp379–396.
`11. Agulhon H: Annales de l’Institut Pasteur, volume 34,
`1910: The presence and utility of boron in plants. Nutr
`Rev 1988;46:353–355.
`12. Dordas C, Chrispeels MJ, Brown PH: Permeability and
`channel-mediated transport of boric acid across mem-
`brane vesicles isolated from squash roots. Plant Physiol
`2000;124:1349–1362.
`13. Bennett A, Rowe RI, Soch N, et al: Boron stimulates
`yeast (Saccharomyces cerevisiae) growth. J Nutr 1999;129:
`2236–2238.
`14. Samman S, Naghii MR, Lyons Wall PM, et al: The nu-
`tritional and metabolic effects of boron in humans and
`animals. Biol Trace Elem Res 1998;66:227–235.
`15. Armstrong TA, Spears JW, Crenshaw TD, et al: Boron
`supplementation of a semipurified diet for weanling
`pigs improves feed efficiency and bone strength char-
`
`American Journal of Therapeutics (2001) 8(5)
`
`FlatWing Ex. 1032, p. 5
`
`

`

`326
`
`GROZIAK
`
`acteristics and alters plasma lipid metabolites. J Nutr
`2000;130:2575–2581.
`16. Naghii MR, Samman S: The role of boron in nutrition
`and metabolism. Prog Food Nutr Sci 1993;17:331–349.
`17. Penland JG: The importance of boron nutrition for
`brain and psychologic function. Biol Trace Elem Res
`1998;66:299–317.
`18. Nielsen FH: The emergence of boron as nutritionally
`important throughout the life cycle. Nutrition 2000;16:
`512–514.
`19. Barr RD, Barton SA, Schull WJ: Boron levels in man:
`Preliminary evidence of genetic regulation and some
`implications for human biology. Med Hypotheses 1996;
`46:286–289.
`20. Naghii MR: The significance of dietary boron, with par-
`ticular reference to athletes. Nutr Health 1999;13:31–37.
`21. Nielsen FH: Ultratrace minerals: mythical elixirs or nu-
`trients of concern? Bol Asoc Med P R 1991;83:131–133.
`22. Nielsen FH: Nutritional requirements for boron, sili-
`con, vanadium, nickel, and arsenic: current knowledge
`and speculation. FASEB J 1991;5:2661–2667.
`23. Nielsen FH: The justification for providing dietary
`guidance for the nutritional intake of boron. Biol Trace
`Elem Res 1998;66:319–330.
`24. Nielsen FH: How should dietary guidance be given for
`mineral elements with beneficial actions or suspected
`of being essential? J Nutr 1996;126:2377S–2385S.
`25. Hunt CD, Stoecker BJ: Deliberations and evaluations of
`the approaches, endpoints and paradigms for boron,
`chromium and fluoride dietary recommendations. J
`Nutr 1996;126:2441S–2451S.
`25A. US Food and Nutrition Board: Dietary reference in-
`takes for vitamin A, vitamin K, arsenic, boron, chro-
`mium, copper, iodine, iron, manganese, molybdenum,
`nickel, silicon, vanadium, and zinc. National Academy
`Press, Washington, DC, 2001, pp13-6–13-14.
`26. Rainey CJ, Nyquist LA, Christensen RE, et al: Daily
`boron intake from the American diet. J Am Diet Assoc
`1999;99:335–340.
`27. Fusayama T: The process and results of revolution in
`dental caries treatment. Int Dent J 1997;47:157–166.
`28. Gaby AR: Natural treatments for osteoarthritis. Altern
`Med Rev 1999;4:330–341.
`29. Clarkson PM, Rawson ES: Nutritional supplements to
`increase muscle mass. Crit Rev Food Sci Nutr 1999;39:
`317–328.
`30. Fail PA, Chapin RE, Price CJ, et al: General, reproduc-
`tive, developmental, and endocrine toxicity of boro-
`nated compounds. Reprod Toxicol 1998;12:1–18.
`31. Richold M: Boron exposure from consumer products.
`Biol Trace Elem Res 1998;66:121–129.
`32. Linden CH, Hall AH, Kulig KW, et al: Acute ingestions
`of boric acid. J Toxicol Clin Toxicol 1986;24:269–279.
`33. Litovitz TL, Klein-Schwartz W, Oderda GM, et al:
`Clinical manifestations of toxicity in a series of 784 bo-
`ric acid ingestions. Am J Emerg Med 1988;6:209–213.
`
`American Journal of Therapeutics (2001) 8(5)
`
`34. Restuccio A, Mortensen ME, Kelley MT: Fatal ingestion
`of boric acid in an adult. Am J Emerg Med 1992;10:545–
`547.
`35. Usuda K, Kono K, Orita Y, et al: Serum and urinary
`boron levels in rats after single administration of so-
`dium tetraborate. Arch Toxicol 1998;72:468–474.
`36. Vaziri ND, Oveisi F, Culver BD, et al: The effect of
`pregnancy on renal clearance of boron in rats given
`boric acid orally. Toxicol Sci 2001;60:257–263.
`37. Pahl MV, Culver BD, Strong PL, et al: The effect of
`pregnancy on renal clearance of boron in humans: a
`study based on normal dietary intake of boron. Toxicol
`Sci 2001;60:252–256.
`38. International Symposium on the Health Effects of Bo-
`ron and its Compounds. Irvine, California. September
`16–17, 1992 (proceedings.). Environ Health Perspect
`1994;102 (Suppl 7):1–141.
`39. Proceedings of the 2nd International Symposium on
`the Health Effects of Boron and its Compounds. Irvine,
`California. October 22–24, 1997. Biol Trace Elem Res
`1998;66:1–473.
`40. Benderdour M, Bui-Van T, Dicko A, et al: In vivo and in
`vitro effects of boron and boronated compounds. J
`Trace Elem Med Biol 1998;12:2–7.
`41. Loomis WD, Durst RW: Chemistry and biology of bo-
`ron. Biofactors 1992;3:229–239.
`42. Antonov VK, Ivanina TV, Berezin IV, et al: n-
`Alkylboronic acids as bifunctional reversible inhibitors
`of ␣-chymotrypsin. FEBS Lett 1970;7:23–25.
`43. Koehler KA, Lienhard GE: 2-Phenylethaneboronic acid,
`a possible transition-state analog for chymotrypsin.
`Biochemistry 1971;10:2477–2483.
`44. Lindquist RN, Terry C, Rawn JD, et al: The binding of
`boronic acids to chymotrypsin. Arch Biochem Biophys
`1974;160:135–144.
`45. Rawn JD, Lienhard GE: The binding of boronic acids to
`chymotrypsin. Biochemistry 1974;13:3124–3130.
`46. Groettrup M, Schmidtke G: Selective proteasome in-
`hibitors: modulators of antigen presentation? Drug Dis-
`cov Today 1999;4:63–71.
`47. Adams J, Behnke M, Chen S, et al: Potent and selective
`inhibitors of the proteasome: dipeptidyl boronic acids.
`Bioorg Med Chem Lett 1998;8:333–338.
`48. Teicher BA, Ara G, Herbst R, et al: The proteasome
`inhibitor PS-341 in cancer therapy. Clin Cancer Res
`1999;5:2638–2645.
`49. Frankel A, Man S, Elliott P, et al: Lack of multicellular
`drug resistance observed in human ovarian and pros-
`tate carcinoma treated with the proteasome inhibitor
`PS-341. Clin Cancer Res 2000;6:3719–3728.
`50. Hideshima T, Richardson P, Chauhan D, et al: The pro-
`teasome inhibitor PS-341 inhibits growth, induces ap-
`optosis, and overcomes drug resistance in human mul-
`tiple myeloma cells. Cancer Res 2001;61:3071–3076.
`51. Cusack JC Jr, Liu R, Houston M, et al: Enhanced che-
`mosensitivity to CPT-11 with proteasome inhibitor PS-
`341: implications for systemic nuclear factor-␬B inhibi-
`tion. Cancer Res 2001;61:3535–3540.
`
`FlatWing Ex. 1032, p. 6
`
`

`

`BORON THERAPEUTICS ON THE HORIZON
`
`327
`
`52. Christie G, Markwell RE, Gray CW, et al: Alzheimer’s
`disease: correlation of the suppression of ␤-amyloid
`peptide secretion from cultured cells with inhibition of
`the chymotrypsin-like activity of the proteasome. J
`Neurochem 1999;73:195–204.
`53. Vacca JP: New advances in the discovery of thrombin
`and factor Xa inhibitors. Curr Opin Chem Biol 2000;4:
`394–400.
`54. Knabb RM, Kettner CA, Timmermans PBMWM, et al:
`In vivo characterization of a new synthetic thrombin
`inhibitor. Thromb Haemostasis 1992;67:56–59.
`55. Kettner C, Knabb RM: Peptide boronic acid inhibitors
`of thrombin. Adv Exp Med Biol 1993;340:109–118.
`56. Lim MSL, Johnston ER, Kettner CA: The solution con-
`formation of (D)Phe-Pro-containing peptides: implica-
`tions on the activity of Ac-(D)Phe-Pro-boroArg-OH, a
`potent thrombin inhibitor. J Med Chem 1993;36:1831–
`1838.
`57. Weber PC, Lee SL, Lewandowski FA, et al: Kinetic and
`crystallographic studies of thrombin with Ac-(D)Phe-
`Pro-boroArg-OH and its lysine, amidine, homolysine,
`and ornithine analogs. Biochemistry 1995;34:3750–3757.
`58. Duncia JV, Santella JB 3rd, Higley CA, et al: Pyrazoles,
`1,2,4-triazoles, and tetrazoles as surrogates for cis-
`amide bonds in boronate ester thrombin inhibitors.
`Bioorg Med Chem Lett 1998;8:775–780.
`59. Skordalakes E, Elgendy S, Goodwin CA, et al: Bifunc-
`tional peptide boronate inhibitors of thrombin: crystal-
`lographic analysis of inhibition enhanced by linkage to
`an exosite 1 binding peptide. Biochemistry 1998;37:
`14420–14427.
`60. Lee SL, Alexander RS, Smallwood A, et al: New inhibi-
`tors of thrombin and other trypsin-like proteases: hy-
`drogen bonding of an aromatic cyano group with a
`backbone amide of the P1 binding site replaces binding
`of a basic side chain. Biochemistry 1997;36:13180–13186.
`61. Kiener PA, Waley SG: Reversible inhibitors of penicil-
`linases. Biochem J 1978;169:197–204.
`62. Beesley T, Gascoyne N, Knott-Hunziker V, et al: The
`inhibition of class C ␤-lactamases by boronic acids. Bio-
`chem J 1983;209:229–233.
`63. Amicosante G, Felici A, Segatore B, et al: Do inert ␤-lac-
`tamase inhibitors act as synergizers of ␤-lactam antibi-
`otics? Utility of boric and boronic acids. J Chemother
`1989;1:394–398.
`64. Crompton IE, Cuthbert BK, Lowe G, et al: ␤-Lactamase
`inhibitors. The inhibition of serine ␤-lactamases by spe-
`cific boronic acids. Biochem J 1988;251:453–459.
`65. Usher KC, Blaszczak LC, Weston GS, et al: Three-
`dimensional structure of AmpC ␤-lactamase from Esch-
`erichia coli bound to a transition-state analogue: pos-
`sible implications for the oxyanion hypothesis and for
`inhibitor design. Biochemistry 1998;37:16082–16092.
`66. Weston GS, Blazquez J, Baquero F, et al: Structure-
`based enhancement of boronic acid-based inhibitors of
`AmpC ␤-lactamase. J Med Chem 1998;41:4577–4586.
`67. Powers RA, Blazquez J, Weston GS, et al: The com-
`plexed structure and antimicrobial activity of a non-␤-
`
`lactam inhibitor of AmpC ␤-lactamase. Protein Sci
`1999;8:2330–2337.
`68. Martin R, Jones JB: Rational design and synthesis of a
`highly effective transition state analog inhibitor of the
`RTEM-1 ␤-lactamase. Tetrahedron Lett 1995;36:8399–
`8402.
`69. Strynadka NCJ, Martin R, Jensen SE, et al: Structure-
`based design of a potent transition state analogue for
`TEM-1 ␤-lactamase. Nat Struct Biol 1996;3:688–695.
`70. Ness S, Martin R, Kindler AM, et al: Structure-based
`design guides the improved efficacy of deacylation
`transition state analogue inhibitors of TEM-1 ␤-lacta-
`mase. Biochemistry 2000;39:5312–5321.
`71. Curley K, Pratt RF: Effectiveness of tetrahedral adducts
`as transition-state analogs and inhibitors of the class C
`␤-lactamase of Enterobacter cloacae P99. J Am Chem Soc
`1997;119:1529–1538.
`72. Coutts SJ, Kelly TA, Snow RJ, et al: Structure-activity
`relationships of boronic acid inhibitors of dipeptidyl
`peptidase IV. 1. Variation of the P2 position of Xaa-
`boroPro dipeptides. J Med Chem 1996;39:2087–2094.
`73. Subrama