Metal-ligand complexes involved in rheumatoid arthritis—I

.L inorg, nucl. Chem. Vol. 40, pp. 118%1194 © Pergamon Press Ltd.. 1978. Printed in Great Britain

0022-190217S10601-11891502.0010

Bio-Inorganic Section

METAL-LIGAND COMPLEXES INVOLVED IN RHEUMATOID ARTHRITIS--I JUSTIFICATIONS

FOR COPPER ADMINISTRATIONt

GRAHAM E. JACKSON, PETER M. MAY and DAVID R. WILLIAMS* Department of Chemistry, University of Wales Institute of Science and Technology, Cardiff, CFI 3NU, U.K. (Received 22 February 1977; received [or publication 17 October 1977)

Abstract--The role of copper in rheumatoid arthritis is discussed both from historical and experimental viewpoints. Reasons supporting the hypothesis that the administration of low molecular weight complexes of copper is beneficial are listed and supported with experimental evidence: (a) Calculations of the bioavailability, in terms of cell membrane transmission, of a series of copper amino-acid mixtures are compared with animal screen results of the effect of such mixtures upon rat paw oedema caused by A carrageenan injection. For subcutaneous administration, the degree of antiinflammatory activity is directly proportional to the quantity of copper injected. (b) The concentrations of ternary copper complexes in serum can be correlated with their protective influence upon the denaturation of serum proteins. The predominant complexes affording protection are copper bistidinate.cystinate and copper histidinate.cystinate.H. INTRODUCTION Rheumatism is a term used to describe painful disorders of the joints or muscles not directly due to infection or injury. Rheumatoid arthritis is one such condition, being a chronic disease of the connective tissue. In the United Kingdom at least 5% of the population are affected by this disease to a greater or lesser degree. Most cases appear as small nodules of inflamed fibrous tissues around the joints especially the knuckles and wrists. Hypotheses concerning the origin of rheumatoid arthritis that are currently in vogue favour disorders of the subject's autoimmune system[l]. It is conceivable that stress (physical, emotional, hormonal, environmental etc.) stimulates an inflammatory response which then overcorrects because the usual feedback mechanism fails to switch off this response• Cures are not likely to become available until far more is known about the causes• However, the pain and inflammation can be alleviated using immunosuppressive agents, analgesics, salicylates and other acid drugs, and gold or copper compounds. Copper was shown to be essential for animal nutrition in 1928. It is present in the blood of humans, a normal adult containing about 76 ms, the average serum concentrations being 1.1-2.0x 10 5moldm -3, females having higher values than males and the concentrations varying slightly with circadian and menstrual cycles[2-6]. The free cupric ion is effectively absent from plasma (estimates of its concentration vary from 10-1' to 10-18 mol dm -3) and so the copper is distributed between two types of complexes--those involving labile and those involving inert bonds. The latter portion of copper is mainly in the form of an alpha2 macroglobulin called ceruloplasmin (this has 6-8 copper atoms per protein *Author for correspondence. tResearch performed at the University of St. Andrews.

molecule and accounts for more than 90% of the copper in plasma). The remaining copper (approx. 10-6 mol dm -3) is present as labile bonded complexes of serum albumin (MW - 67000, containing only one copper binding site of any physiological importance) and of low molecular weight ligands (such as the anions of aminoacids). The concentrations of individual copper complexes in the exchangeable portion of plasma (i.e. the serum albumin and especially the low molecular weight components) cannot be monitored by any known instrumental techniques. However we have recently reported a computer simulated model of several thousand low molecular weight complexes in plasma and found that the metal ion exists as the following predominant complexes--Cu.histidinate.cystinate-, Cu.histidinate.cystinate.H °, Cu.histidinate~, and Cu.histidinite.threoninate°.[7]. This circulating pool of low molecular weight copper is the form in which copper is donated into tissues; cell membranes can be traversed by those low molecular weight complexes which are neutral because they are soluble in the lipoprotein matrix. The total concentrations of metal present are, of course, amenable to measurement using conventional techniques such as atomic absorption spectrophotometry. Niedermeir has reported analyses for some 14 elements in the blood and synovial fluids of normal, arthritic, and gold-treated patients, total copper being high in both fluids when rheumatoid arthritis is present[8--10]. However there are disparate opinions concerning which component is responsible for this elevated concentration: Niedermeir et al. claim that ceruIoplasmin copper is raised [10] whereas Lorber et al. find that it is non-ceruloplasmin bound copper[Ill that exhibits an increased concentration. Sternlieb et al. favour the former opinion [ 12]. It is interesting that inflammatory

1189

1190

G. E. JACKSON et al.

states in general, and joint destruction diseases in particular (e.g. ankylosing spondylitis), also exhibit elevated blood copper levels [13, 14]. Our aims are (i) to list the somewhat circumstantial evidence that copper is involved in rheumatoid arthritis, (ii) to examine literature reports that low molecular weight complexes of copper participate, and (iii) to discuss data for Sorenson [15,16], Gerber[17-20] and ourselves which support this hypothesis. LOW MOLECULARWEIGHTSPECIES Folk-lore has treated rheumatoid arthritis with copper rich remedies for many centuries (shell fish, nuts, cider vinegar and copper jewellery[3]). More recently there have been researches aimed at quantifying the effect of dermal assimilation of copper from bracelets worn in arthritic conditions and investigations of the copper dependency of the synthesis of prostagladins involved in producing and in reducing inflammation[21, 22]. Evidence that low molecular weight complexes of copper are at the heart of the rheumatoid arthritis therapy is accumulating and is very persuasive. Walker et al. have recently separated plasma into different molecular weight fractions and shown that species of molecular weights below 500 contain chemicals active against rheumatoid arthritis [23]. This fraction was found to release chemotactic factors, a process in which histamines, tryptamines, kinins and prostaglandins are apparently not involved. Such an observation is difficult to reconcile with the report that one's natural immune response involves the formation of a binuclear hydroxybridged complex of copper and histamine, H [ h m . C u ( : ) C u . h m ] 2+ the formation of which is H antagonised by the familiar application of zinc compounds to treat allergies[24]. Indeed, binuclear copper complexes of low molecular weight which are postulated to exist in vivo ought generally to be regarded with suspicion in view of the very high ligand to metal ratios found in all biofluids. Copper is specifically implicated in a number of possible mechanisms of inflammation and its arrest. McCord has suggested that superoxide radicals are involved in initiating inflammation[25] and it has subsequently been postulated that free radical scavengers (for example bis(salicylato)copper(II) or the copper enzyme superoxide dismutase) protect the synovial fluid from hydroxyl and superoxide radical attack[26-28]. Even the widespread hypotheses based upon prostaglandins can encompass a role for copper [22, 29]. The normal endogenous response to inflammation has been postulated to involve low molecular weight species which become firmly bonded to serum albumin[23,30]. Further, administered acidic drugs also bind to albumin. (We have investigated this aspect in Part II.) The antiarthritic activity of these endogenous and exogenous agents we suggest is through a mechanism which encourages the release of albumin-bound copper. Another low molecular weight drug, l>-penicillamine, has

also been used in treating rheumatoid arthritis[31,32]. Frequently it is capable of effecting remissions and we have suggested that it labilizes body stores of copper into low molecular weight form, ceruloplasmin bound copper being a possible source[33]. However, there are many mysteries associated with l>-penicillamine therapy: (a) L-penicillamine is claimed to be ineffective (the low LDso may be due, however, to the toxicity of this isomer rather than to less therapeutic activity), (b) patients take several weeks to respond to l>penicfflamine therapy[34], and (c) some patients do not respond at all [34-37]. The effect of D-penicillamine on connective tissue metabolism has received considerable attention, often in the context of rheumatoid inflammation[38]. Although various mechanisms involving sulphydryl interactions have been postulated[38, 39] interference with the transition metals required for collagen synthesis (see Part IV) is an attractive possibility. A viable alternative to the use of endogenous copper is one of copper administration. There are two convenient routes--dermal and oral. Recent studies in Australia[21,40] have shown that a copper bangle can dissolve in human perspiration to the extent of l0 -3 tool dm -3, a typical weighed bracelet losing 1 mg per day or more. Our normal daffy intake of copper is 2.5-5.0mg, approx. 30% being absorbed in the small intestine as low molecular weight complexes of monomeric, acidic amino-acids of the L-configuration. However, other metals and ligands (e.g. Ca 2+ or P O t - ) suppress this absorption. Unfortunately non-absorbed orally administered copper salts are highly irritant causing ulceration and oedema[16, 41]. Thus, it would appear desirable to complex the copper in less irritant complexes and to select compounds that optimise the degree of dermal or intestinal absorption. (This aspect is researched further in Part VI.) Other trace elements used in the treatment of rheumatoid arthritis include calcium (mainly in the form of calcium Aspirin), zinc[42] and gold (mainly as Au(I) complexes of sulphur donor ligands such as thiomalate, thioglucose, thiopropanol or thiosulphate; Au(O) colloids and Au(III) chloride have also been used). A large proportion of this gold eventually complexes onto the serum albumin in plasma and perhaps it has similar roles to those of copper. However, since gold is not normally found in humans this series of researches has concentrated on copper. DATA FROM LABORATORY AND

ANIMALEXPERIMENTS We shall consider the influence of copper complexes of established drugs for arthritis, and then in vivo and in vitro experiments involving copper complexes of naturally occurring ligands. Quantitative observations concerning the antiinflammatory role of copper-drug compounds have been made by Sorenson who reported that salts of many acidic antiiflammatory drugs currently in use are more effective at reducing inflammation than their parent acids. (The complete verification of this principle was extremely difficult since the copper salts were usually too insoluble to administer[15, 16].) An additional advantage of these copper complexes was that their antiulcer activity was superior to that of the parent acid. In a later part of this series we have interpreted these biological response data in terms of our computed concentration activity relationships (see Part VI).

Metal-ligandcomplexes involved in rheumatoid arthritis--I

1191

The experimental investigations conducted in our such as albumin, the biological response data ought to be laboratory were aimed, in the main, at copper complexes directly proportional to the total copper content of the of naturally occurring ligands such as amino-acids rather complexes administered regardless of speciation. Indeed than already established rheumatoid arthritis drugs. This model studies (in Part VI) show that, in general, all low was decided in order to reveal the effect of copper molecular weight copper complexes dissociate when insupplementation without the assistance of exogenous jected into plasma. Alternatively, if it is necessary for chemicals, i.e. to see whether the normal antiinflam- some complexes to pass through cell membranes into matory response of animals benefitted from administered another media then the activity ought to reflect the copper. We also introduced another simplification concentration of neutral complexes present under phycompared to the Sorenson data in that the problems of siological conditions. To achieve this latter purpose it is oral absorption were circumvented by using sub- possible to choose a wide spectrum of ligands ranging cutaneous administration as a means of administering from good promoters of copper, in terms of membrane complexes slowly, but directly, into plasma without hav- solubility, to poor ones. The range of ligands considered, ing to consider bioavailability and absorption their formation constants and the computed concentrations of complexes formed are shown in Table I. This phenomena. All low molecular weight complexes considered in this suggested a membrane penetration order of Cu(aand subsequent papers are considered to have labile aminobutyrateh > Cu(phenylalanateh > CU(DL-4- amino copper(lI)--ligand bonds and so mixtures of cupric - 3 - hydroxybutanoate)2 ,> Cu(tyrosinateh > Cu(lysinateh chloride and ligands were employed. This is at variance > CuCI2. These representative species (along with gluwith other workers who have synthesised copper tathionate and Aspirin to be researched in later parts of the chelates for administration[15, 16, 43]. We believe it is a series) were then screened. The test used was the percentage inhibition of an truism that whether a biosolution is prepared from presynthesized copper chelates or from mixtures of induced oedema formed in the rat paw by an injection of metal salt plus ligand, provided the same total concen- X-carrageenan. The mixtures were administered subtrations of copper and ligand are used in both instances, cutaneously, in the doses indicated in Table 2. one hour the equilibrium distribution of copper complexes prior to the carrageenan injection and the amount of produced upon administration will be exactly the same oedema was measured three hours later. The resulls are (i.e. unless kinetically inert complexes such as porphy- listed in Table 2. The number of mg/kg administered were so arranged to give equal molar doses of copper rins are administered). Thus mixtures of copper chloride and ligands were and it is clear that all the lower doses given the same injected into animals subcutaneously. The ligands were percentage inhibition range and that the higher doses all chosen in the light of COMPLOT computer model give the same order of effectiveness which is apstudies of the equilibria occurring at physiological pHs proximately double the lower figure. This suggests (a) [44, 46J. In principle this involved selecting a standard that the protection against oedema afforded by the mixdose and using this as computer input data, along with tures is directly proportional to the total amount of the formation constants for ligand-proton, ligand-copper copper administered, (b) that cell membrane transmission and copper-hydroxo formation reactions, and so of the intact complexes as injected is not involved, (c} computing the distribution of species in aqueous solution that all of the copper complexes administered merely at various pH values (an example is shown in Fig. 1). supplement the metal and ligand components of normal We reasoned that if the complexes were merely adding rat plasma, and (d) that the mechanism controlling the copper directly to the antiinflammatory process in oedema is capable of aquiring the administered copper plasma and if this mechanism involved a powerful ligand ions regardless of whether it is relatively firmly

15

10

--

% x

o5

CuA

HA-

o

I

2

3

4

L

5

6

7

8

9

K)

pH

Fig. 1. COMPLOT calculation of complexes present in a solution of copper chloride (7.50x 10-4 tooldm-3) and DL-4-amino-3-hydroxybutanoicacid. As- represents the ligandanion.

i192

G.E. JACKSON et al. Table 1. Input data for the COMPLOT models. Total concentrations (moldm-3) were copper = 7.50x 10-4, ligand = 1.50x 10-~. The computed concentration of each species at plasma pH is also shown Complex composition Ligand

p,

q

r

Histidilmte

1

1

'0

2

1

o*

t

Concentration of complex at log Ppqr

• I=7.4

-6

Lysinate

i0.086 a

4.64 x 10

19 .O26

5 . 5 9 x 10

6.25 x i0-8 -4 1.60 x 10 -7 4 , 6 9 x 10 -5 2.77 x i0

1

1

1

15.615

2

1

1

25.884

2

1

2

30.750

1

1

1

1

2 2

3.462

"1 1

17.910

1

2

34.770

1

1

25.870

2

1

O*

14.600

1

1

0

b

-4

8 . 8 9 x i0

-5

6 , 4 0 x 10- 4 -5 2.02 x 10 -9 2 . 7 3 x 10 -5

a-Aminobutyrate

2 Asparaginate

Phenylalartate

Tyrosinate

Triethylenetetramine

Oxidised penicillaminAte

D L - 4 --Amino-3 - h y d r o x y butyrate

Footnotes (i) (ii)

Superscript

*

1

7.650 b

o*

15.090 7.690

I

1

O

~.

1

o*

1

1

0

2

1

o*

1

1

O

2

1

o*

b

b

7.08

-4 x i0

i. 16 x iO

-4

6 . 1 1 x i0 -5 -4

6 . 8 8 x 10

14.410 9.320

x 10

6.33 x 1 0 - 4

13.660 7.650

4.10

b

7.12 x i0

-6

-8

15.090

1 . 8 9 X i0

6 . 4 9 x 10 - 5 -4 6.70 x IO

1

1

1

17.680

2

1

2

34.440

2

1

1

25.080 b

7.34

x 1 0 ~6

7.50

x 10-4

1

1

0

19.410

1

1

2

24. 950

1

1

1

22.030

1

1

o*

2

2

O*

27.400

3.75

1

1

1

15.510

4.98 x i0

4.12 x 10"13 -8 1.25 x I0 b

7.220

1

1

z

2

o* o*

28.696

2

1

0

18.856

6.41 x 10 " 9 x 10 "4 -8

4.76 x 1 0 . 4

13.961 c

1.35 x 10 .4 5.75

x 10

-9

denotes membrane soluble neutral complex

refers to the composition of the general complex (ligand)p(copper lOn)q(proton) r. Negative values of r refer to

the nt~Rber of hydroxide lops involved References to data used:a b

D.R. Williams, J. Chem. 8oc. Dalton, 1972, 790. D.D. Perrin and R.P. Agarwal,

"Metal Ions in Biological Systems" Ed

H. Slgel, 1973, ~, 167. A. Braibanti, G. Morl and E. Lepor~ti, J. Chem. Soc. Dalton, 1975,

complexed (as with copper-a-aminobutyrate) or only rather weakly complexed (e.g. cupric chloride). This implies that the binding of copper ions to endogenous ligands in tissues is powerful which is promising from the point of view of other complexes being screened. (There have been instances of metal supplementing complexes being well absorbed from the intestine but being unable to release their metal ions to the body's biochemistry. For example, zinc histidinate is well absorbed hut rapidly excreted [47].) Oral administration of the mixtures listed in Table 2,

1319.

with the exception of Aspirin-Cu, gave no apparent protection against oedema. This aspect was not rigorously pursued in the animal experiments and so the data are not tabulated. We decided to delay investigations into means of promoting oral absorption until we had illuminated as many aspects as possible concerning the serum biochemistry of pharmacoactive copper complexes with the objective of optimising the desirable properties whilst minimising the side effects (see later parts of this series). In all instances where equivalent doses of copper

Metal-ligandcomplexes involved in rheumatoid arthritis--i

1193

Table 2. Screeningresults for copper mixtures administeredto carrageenan induced oedema model. The doses are arranged to give equivalent doses of copper Substance screened

%Cu

Cu: (glutathionate) 2

8. i

Cu: (lysinate) 2

ii .8

Cu~tyros inate)2

i i. 9

Cu~acety isa licy late)2

Cu: (phenyla laninate) 2

Cu: (DL-4-Amino-3-hydroxybutanate)2

Cu: ( u - a m i n o b u t y r a t e )

Cu: (chlorlde) 2

2

11.9

12.7

15.5

16.9

37.3

Dose (mg/kg)

% inhibition o f oedema f o r m a t ion.

115

50.8

46

22 .9

79

82.4

32

34.3

78

44 . 7

31.3

20.6

78

54 .5

31

13.9

73

56,9

29.4

12.9

60

66.4

24

19 .~

55

58.1

22.1

41.9

25

46.4

i0

32

complexes and copper chloride-parent acid mixtures genesis of the disease, ternary amino-acid copper were administered subcutaneously the same percentage complexes protecting plasma proteins against denaturaprotection against oedema results were obtained and the tion. Without such protection aggregated v-globulins act same degree of central nervous system toxicity side as antigens stimulating the production of rheumatoid effects noted. factors and inflammation[19, 20]. Gerber has reported the inhibitory effect in vitro of a These results are clearly related to the observations variety of naturally occurring amino-acids and metal ions that blood histidine levels of rheumatoid arthritic upon the sulphydryl-disulphide interchange-mediated patients are 1ow[17-19], and that the activity of the denaturation of human gamma globulin, bovine serum disease is proportional to the magnitude of the aminoalbumin and diluted human serum. Data are reported in acid deficiency. The inflammation does indeed respond to terms of solubilities and viscosities. We have been able oral administration of L-histidine [20,481. to assess some of the data in their chemical context and CONDLUCING REMARKS study species at the molecular level by using computer simulation of the solution studies as computed by the The most relevant findings of this study are (i) that ECCLES program [7]. copper ions per se can afford protection against inflamThe amino-acids alanine, arginine, aspartic acid, gly- mation even without the assistance of administered cine, leucine, phenylalanine, proline, serine, threonine, organic therapeuticals, (ii) it seems relevant to monitor tyrosine and salicylic and uric acids did not confer plasma copper, histidine and cystine contents of arthritic protection against denaturation. Similarily, calcium, iron, patients and to supplement if necessary, and (iii) low magnesium sodium, nickel and zinc were inactive. molecular weight copper in plasma occurs predominantly Significant protection was afforded by histidine, cystine as ternary complexes all of which involve histidinate and and copper, individually or administered together. the most important having cystinate as the secontlary Preliminary results of the ECCLES simulations of ligand. The existence of these species can account for solutions of protein and diluted serum to which the both of the foregoing conclusions. ligands and metal ions above had been administered demonstrated that the trends in Gerber's in vitro data Acknowledgements--Two of us (P. M. M. and G..E.J.) thank the can be correlated directly with the concentrations of C.S.I.R. (R.S.A.) C. J. Adams and Leverhulme Trusts for mainCu.histidinate.cystinate- and Cu.histidinate.cystinate.H tenance grants. We also thank Dr. F. Mangan of Beecham Pharmaceuticalsfor complexes. Furthermore it appears that variations in the total copper concentration, the total low molecular conducting the animal screening experiments. weight copper concentration or the concentration of free copper ions do not follow the patterns of data displayed REFERENCES in Ref. (19). 1. Medical News, Postgraduate Series on Rheumatism. Tribune In Part VI we discuss in vivo evidence that such Press, London, Nov.-Dec. (1976). ternary complexes of copper are important in the control 2. E. B. Hart, H. Steenbock,J. Waddelland C. A. Elvehjem.J. of inflammation and the mechanisms through which Biol. Chem. 77,777 (1928). copper and amino-acids are transfered through 3. M. W.,Whitehouse,Agents and Actions 6, 201 (1976). membranes to tissues or into synovial fluid. Gerber has 4. G. S. Shields, H. Markowitz, G. E. Cartwright and M. M. suggested that subnormal histidine and copper concenWintrobe, Metal Binding in Medicine (Edited by M. J. Seven trations in synovial fluid contribute towards the pathoand L. A. Johnson), p. 259. Lippincott, Philadelphia(1960).

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G.E. JACKSON et al.

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