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Pharmacologic actions of 4-aminoquinoline compounds
Pharmacologic Actions of 4-Aminoquinoline Compounds
ALLEN H. MACKENZIE, M.D. Cleveland, Ohio
The pharmacokinetics, physiologic effects, and the metabolization of chloroquine and hydroxychloroquine are all similar. Their concentrations in plasma and tissue are directly related to dally dosing. The highest concentrations are found in melanin-containing tissues, particularly the choroid and ciliary body of the eye. The pharmacologic effects of 4-aminoquinoline compounds are reviewed in detail. It is likely that the rheumatologic effectiveness of these agents is primarily related to lysosomal actions. The drug-induced lysosomal abnormalities include diminished vesicle fusion, diminished exocytosis, and reversible “lysosomal storage disease.” It is likely that the retinal toxicity of these drugs is one manifestation of the altered lysosomal physiology involving the retinal pigmented epithelium. Tissue of retinal pigmented epithelium is similar to that of the bone-marrow-derived macrophage. Depression of extra-oculogram is an early sign of excessive dosage and can be used to measure potential toxicity during therapy with 4-aminoquinolines. Dosages ranging from 3.5 to 4.0 mg/kg per day for chloroquine and 6.0 to 6.5 mg/kg per day for hydroxychloroqulne are clinically safe. The beneficial effects of 4-aminoquinoline compounds in the management of rheumatoid arthritis, systemic lupus erythematosus (SLE), and other connective tissue diseases were discovered through serendipity [ 1,2]. While subsequent controlled studies have amply demonstrated the efficacy of these compounds in treating rheumatoid arthritis [3-111, and have established a level of efficacy for the management of SLE, use in juvenile rheumatoid arthritis [2,12] is investigational. They should only be used under approved protocols by experienced clinicians. METABOLISM
From the Department of Rheumatic and Immunologic Disease, Cleveland Clinic Foundation, Cleveland, Ohio. Requests for reprints should be addressed to Dr. Allen H. Mackenzie, Department of Rheumatic and Immunologic Disease, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44106.
Currently, the two most used 4-aminoquinoline compounds are chloroquine phosphate, more popular in Europe [ 13,141, and hydroxychloroquine sulfate, more popular in the United States [ 15-l 7] _ These two agents are very similar antimalarials, differing only in a single hydroxyl group at the end of the side chain. Both are also similar in kinetics [ 181, actions [ 1,191, and metabolism [20,21]. I will therefore treat them alike; what I say of one, largely applies to the other. These basic amines have a heterocyclic planar rl-aminoquinoline nucleus at one end of the polar molecule and a lipophilic side chain on the other end [22], and their kinetics have been fairly well studied [ 181. They are rapidly and completely absorbed when given by mouth [21], and approximately 50 percent are transported by binding to serum proteins [23,24]. Their elimination proceeds in two stages. In the first stage there is rapid excretion with a half-time of
July 18, 1983
The American Journal of Medicine
5
PLAQuENILSYMPOS~UM--MACKENZIE
EXCRETION
INTESTINE
5%
’
and spleen; yet higher concentrations occur in kidney, bone marrow, lungs, and liver, while even higher concentrations are found in the adrenal glands and pituitary and the highest of all occur in melanin-containing tissues, particularly the choroid and ciliary body of the eye [20,28-301. Concentrations several hundred times those in the serum are found in liver, where an approximately 1 X 10e4 M concentration is found at therapeutic serum concentration (1 X lo+ M) [ 19,3 l-331. The therapeutic serum concentrations attainable in man at a clinically safe dosage of chloroquine (3.5 to 4.0 mg/kg per day) [34], produces serum levels of 0.6 to 0.9 pmol/L 119,311. For hydroxychloroquine, safe daily dosage (6 to 6.5 mg/kg per day) 1341 produces serum levels of 1.4 to 1.5 pmol/L [ 19,311. PHARMACOLOGIC
I...
___
Fbure 1.
Diagram of the kinetics, distribution, therapeutic tissue concentration levels, anddisposition of hydroxychloroquine in man 1331.
about three days. The second stage has a more prolonged half-time of about 18 days. Clinical half-life, on the other hand, is 50 to 52 hours for each agent, rising to a higher value at very high serum concentrations [ 181 (Figure 1). About 50 percent of the administered dose has been identifiably recovered [21]. Thus, of the identifiable 4-aminoquinoline compounds excreted, 50 to 60 percent is in the urine, while 8 to 10 percent of chloroquine and up to 15 to 24 percent of hydroxychloroquine is fecally excreted [ 251. Hydroxychloroquine is reportedly conjugated with glucuronide and excreted in the bile [ 20,211. Unknown amounts of 4-aminoquinoline compounds are deposited into dermal cells and appendages, and sloughed off by the skin [ 261. The remainder is thought to be metabolically biotransformed to molecules not readily perceived as being derived from 4-aminoquinoline compounds [ 201. The 4-aminoquinoline compounds attain plasma concentrations that are directly related to the magnitude of the daily dosage [ 231 in a clear-cut, dose-response relationship (Figure 2). At a given serum concentration, the drug is distributed throughout various body tissues, depending upon tissue affinity for the compound [ 271. Fat, bone, tendon, and brain contain relatively small amounts of 4-aminoquinolines [ 201. Lowest tissue concentrations are found in skeletal muscle, skin, and sclera; the next higher concentrations are found in heart
6
July 16, 1963
The Am&can
Journal of Medicine
EFFECTS
Although a broad spectrum of biologic actions of 4aminoquinolines is demonstrable in vitro. or in animals at higher concentrations, these effects are not clinically valuable because they require dosage levels that are too toxic to permit prolonged therapy. Thus, the actions of antimalarial drugs at the above-mentioned.concentrations are the only effects safely attainable in vivo [ 11. The spectrum of pharmacologic actions that pertain in rheumatology probably span the concentration range from 10m7 M to 10m4 M in tissues, but never higher than about 1.5 X 10m6 M in serum. In this concentration range, three principal groups of effects are observable: 1. The 4-aminoquinolines form a complex with ferriprotoporphyrin IX, an intermediary product of hemoglobin digestion by malarial parasites (plasmodia) (351. This complex is so toxic that ions are lost from the parasite cells, osmotic lysis occurs, and malaria is inhibited or cured. Plasmodia resistant to the chloroquine action digest little hemoglobin. Such resistant plasmodia appear no more susceptible to chloroquine than are the cells of the host. 2. Chloroquine influences the behavior of lysosomes, interfering with the vesicle fusion process in a cell [36,37]. Lysosomes increase in number, increase in volume, decrease in density, and develop lamellar membrane structures called myeloid (or myelin) bodies [38-411. Such myelin bodies are thought to be phospholipid configurations [37,41] in phagocytic or autophagic vacuoles. Treated cells are unable to proceed at normal rates with orderly pinocytosis 1421, exoplasmosis, and phagolysosomal fusion [43], with the result that large quantities of cellular membrane are sequestered within the cell in the increased number of lysosomal vesicles. These vesicles contain or consist of plasma membrane phospholipids 1411, with attached cell receptors [ 421. This population of vesicles normally
PLAQUENIL SYMPOSIUM-MACKENZIE
10-5M
500 mg/d
(13 3mg/Kg/d)
10e6M
250mg/d
(4 mg/Kg/d)
/
10e7M 500mghk
Figure 2. A family of curves showing the time course to attainment of plateau concentrations of chloroquine in plasma, and the dependence of plateau plasma concentration on daily dosage rate. While these curves describe the dynamics of chloroquine, hydroxychioroquine behaves in much the same way [d-6,25,26].
(I 2mg/Kg/d (0 6mg/Kg/d
0
eqwalent) equwalent)
2
I MONTHS
I
recycles intact units of membrane rapidly from the surface of the cell into pinocytic and phagosomal vesicles, to the endoplasmic reticulum and golgi, thence back to the cell surface. Primary lysosomes possess a membrane composition similar to that of the plasma membrane [44]. The entire cell surface membrane of a working phagocyte of the macrophage type is recycled approximately every five minutes [42]. A number of specific absorptive endocytosis systems are thought to require this membrane with its receptors in order to operate efficiently. Sequestration of so much plasma membrane in the stabilized vesicles depletes the cell surface of receptors for all manner of molecules, because these receptor sites are imbedded into the phospholipid plasma membrane [42,43]. The depletion of cell surface receptors probably diminishes the rate or efficiency with which a cell responds to its environment. This has been well proven to be true for acid hydrolases [42] of several types, but probably applies also to other receptors, for example, those for phytohemagglutinin. Phytohemagglutinin responsiveness is diminished in chloroquine-treated cells [45,46]. Thymidine uptake has been shown to be diminished. However, the 4-aminoquinoline compounds do not affect receptor affinity [42]. Depletion of cell surface receptors caused by reversible inhibition of membrane recycling appears to be the mechanism, rather than blockade of receptors [43]. Complex functions, such as phagocytosis [47] and chemotaxis [48], are probably dependent on these receptors. Although these effects are completely reversible [42], cellular efficiency is greatly diminished [36]. The growth of cells is inhibited, and a significant amount of cell death will occur at concentrations in excess of therapeutic levels [46]. The mechanism by which the 4-aminoquinoline drugs inhibit vesicle fusion is not yet clear. Some data suggest a chloroquine/clathrin interaction in which the latter is solubilized [49]. Clathrin is a structural protein, coating vesicle walls, which is probably involved in vesicle fusion [44,50,51]. The properties of clathrin are
altered by lysosomotropic amines, elevated pH, and other factors [49]. 3. In the presence of chloroquine or hydroxychloroquine, the digestive efficiency of phagolysosomes is diminished [38,52,53]. Studies have shown delayed digestion during cellular autophagy in the presence of 4-aminoquinoline drugs for several differing constituents: (1) some cellular proteins, (2) mucopolysaccharides, (3) hormones, and (4) phospholipids [53]. The controlled autophagy necessary to the early stages of derepression in a transforming lymphocyte [46,52] or in a cell preparing to undergo mitosis is delayed. The basic amine molecules become concentrated in the lysosomes. The drug passively diffuses into the cell in a lipophilic basic unprotonated state, but once it enters the acid milieu of a lysosome (pH about 4.0), the 4aminoquinoline compound becomes progressively protonated [49]. Protonation renders the side chain more lipophobic, and hence traps the drug within the lysosome. The entry into the cell is by passive diffusion, not requiring energy, but the trapping mechanism depends upon the proton pump in the wall of the lysosome, which does require energy. The pH of a chloroquinetreated lysosome rises from about 4.0 or 4.5 to approximately 6.0 pH units [54]. It is not certain how hydroxychloroquine and chloroquine interfere with lysosomal digestion. Since the pH rises within the lysosome, it conceivably could rise enough to exceed the optimal range for acid hydrolases to perform their digestive functions. In addition, the dissociation of the lysosomal hydrolases from their mannosyl recognition marker “receptor” does not take place with normal speed in the presence of chloroquine [42]. This inhibits the discharge of contained enzymes into a phagosome or to the cell surface. There seems to be a real likelihood that such important cellular activities as feeding, secreting, excreting, synthesis, mitosis, immune transformation, and autophagy [55] depend upon a functional lysosomal system. Thus, the sequestration of receptors within the altered lysosomes could depress immune
July 18, 1883
The American Journal of Medicine
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PLAQUENIL SYMPOSIUM-MACKENZIE
responsiveness by depletion of appropriate receptors. Add to this the delayed vesicle fusion and inhibited phagosomal digestion, and the outcome is considerable reversible interference in cell work. 4. The 4-aminoquinoline compounds are bound to numerous tissue constituents such as nucleoproteins [ 561, melanins [ 281, porphyrins [35,57], and the like. This begins to be significant at about 1 X 10m6 M serum concentration and becomes much more intense at higher concentrations. The effect on nucleoprotein alters the physical properties of DNA and RNA, thus delaying excision and repair and diminishing the efficiency of polymerases [ 58-611. Effects attainable at still higher concentrations do not appear to be therapeutically significant. In summary, it appears likely that the rheumatologic effectiveness of the 4-aminoquinoline drugs is related primarily to the lysosomal actions. These take place at concentrations safely attainable in the plasma of patients during therapy. ABNORMAL
LYSOSOMAL
FUNCTIONS
Three 4-aminoquinoline-induced abnormalities of lysosomal function are known to occur: 1. Chloroquine diminishes vesicle fusion, perhaps by modifying clathrin [49]. This sequesters large quantities of plasma membrane in the lysosomes, thus trapping as much as 50 percent of receptors within, away from the cell surface [42]. This would appear to alter the cell’s responsiveness, not only to many ordinary stimuli, but probably also to immune stimuli. 2. Exocytosis of cell products is diminished by the same inhibition of vesicle fusion. Salmeron and Lipsky [62] have found lnterleukin 1 concentration to be greatly diminished in vitro when using very low concentrations of chloroquine. Conceivably, lnterleukin 1 is secreted via exocytosis of transport vesicles, via carrier-receptors, or via both. Cells rich in lysosomes appear particularly susceptible to these effects of the 4-aminoquinoline compounds. Polymorphonuclear leucocytes, macrophages, synovial macrophages, and retinal pigmented epithelium macrophages are all altered in morphology and in biologic responses by 4aminoquinolines in therapeutic concentrations. 3. Lie and Schofield [38] have drawn the analogy of resemblance to a “lysosomal storage disease” during therapy with chloroquine. Storage results when diges-
tive processes cannot proceed to completion, so that residual matter is left in lysosomes/phagosomes unable to be exocytosed. RETINAL
PIGMENTED
EPITHELIUM
Elner et al [63] have put forth evidence that the retinal pigmented epithelium is a tissue very similar in its structure and responses to bone marrow-derived macrophages. The cells of retinal pigmented epithelium possess microvilli and numerous ruffles under electron microscopy, and they demonstrate phagocytosis, glass adherence, and the ability to phagocytose erythrocytes coated with antibodies. It is the duty of the retinal pigmented epithelium to scavenge, to phagocytose, and intracellularly to digest the aged photoreceptor membrane discs in its phagolysosomes. Photoreceptor discs are shed diurnally from the tip of the rod and cone cells under the influence of light. Potts [29] has demonstrated dose-related depression of retinal pigmented epithelium metabolism, which is reversible up to very high drug concentration levels. The photoreceptor lesion appears to be largely secondary to retinal pigmented epithelium depression during 4-aminoquinoline therapy, and it too has been reversible up to the point of extensive cell death, which correlates with excessive daily doses. The biologic current generated in the retinal pigmented epithelium is measured as the extra-oculogram. Depression of the extra-oculogram is an early sign of excessive 4-aminoquinoline dosage, and has been used to monitor eyes during therapy [ 141. It is reasonable to suspect that a dosage-dependent, pharmacologicinduced lysosomal storage disease of the retinal pigmented epithelium might cause retinal dystrophy from inefficient elimination of photoreceptor cell debris. It is also reasonable to suspect that a threshold level of this phenomenon can be defined in terms of dosage or concentration, above which toxicity develops but below which therapy may be conducted in relative safety [34,64,65]. Since the retinal lesion is the only medically significant toxicity of antimalarial drugs [66], all other adverse effects being reversible, studies were undertaken to attempt to define a therapeutically safe zone, and the threshold of toxicity. These will be discussed in my paper, “Dose Refinements in Long-Term Therapy of Rheumatoid Arthritis with Antimalarial%” appearing elsewhere in this symposium issue.
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Mackenzie AH, Scherbel AL: Chloroquine and hydroxychloroquine in rheumatological therapy. Clin Rheum Dis 1980; 6(3): 545-566. Dub&s EL: Antimalarials in the management of discoid and systemic lupus erythematosus. Semin Arthritis Rheum 1978; 8(l): 33-51.
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The American Journal of Medicine
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Dwosh IL, Stein HB, Urowitz MB, Smythe A, Hunter T. Ogryzlo MA: Azathioprine in early rheumatoid arthritis. Comparison with gold and chloroquine. Arthritis Rheum 1977; 20(2): 685-692. Freedman A, Steinberg VL: Chloroquine in rheumatoid arthritis. A double blindfold trial of treatment for one year. Ann
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Gerber DA: Effect of chloroquine on the sulfhydryl group and the denaturation of bovine serum albumin. Arthritis Rheum 1964; 7(3): 193-200. Zvaifler NJ, Rubin M, Bernstein H: Chloroquine metabolism--drug excretion and tissue deposition [abstr]. Arthritis Rheum 1963; 6: 799-800. Shaffer B, Chan MM, Levy EJ: Absorption of antimalarial drugs in human skin. J Invest Dermatol 1958; 30: 341-345. Rubin M: The antimalarials and the tranquilizers. Dis Nerv Syst (Suppl) 1968; 29: 67-76. Potts AM: The reaction of uveal pigment in vitro with polycyclic
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compounds. Invest Ophthalmol 1964; 3: 405-416. Potts AM: Further studies concerning the accumulation of polycyclic compounds on uveal melanin. Invest Ophthalmol 1964; 3: 399-404. Larsson B, Tjalve H: Studies on the mechanism of drug-binding to melanin. Biochem Pharmacol 1979; 28: 1181-l 187. Wollheim FA, Hanson A, Laurel1 CB: Chloroquine treatment in rheumatoid arthritis. Stand J Rheumatol 1978; 7: 161-176. Alving AS, Eichelberger L, Craige B Jr, Jones R Jr, Whorton CM. Pullman TN: Studies on the chronic toxicity of chloroquine. J Clin invest 1948; 27(suppl): 60-65. McChesney EW, Banks WF Jr, McAuliff JP: Laboratory studies of the 4-aminoquinoline antimalarials: II. Plasma levels of chloroquine and hydroxychloroquine in man after various oral dosage regimens. Antibiot Chemother 1962; 12: 583594. Mackenzie AH: Ocular safety of huge cumulative antimalarial dosage [abstr]. Arthritis Rheum 1981; 24(suppl): S70. Chou AC, Fitch CD: Hemolysis of mouse erythrocytes by ferriprotoporphyrin IX and chloroquine: chemotherapeutic implications. J Clin Invest 1980; 66: 856-858. DeDuve C, DeBarsy T, Poole B, Trouet A, Tulkens P. Van Hoof F: Commentary-lysosomotropic agents. Biochem Pharmacol 1974; 23: 2495-2531. Harder A, Kovatchev S, Debuch H: Interactions of chloroquine with different glycerophospholipids. Hoppe-Seyler’s Z Physiol Chem 1980; 361: 1847-1850. Lie SO, Schofield B: Inactivation of lysosomal function in normal cultured human fibroblasts by chloroquine. Biochem Pharmacol 1973; 22: 3109-3114. Fedorko M: Effect of chloroquine on morphology of cytoplasmic granules in maturing human leukocytes-an ultrastructural study. J Clin Invest 1967; 46(12): 19321942. Datsis AG: Biochemical lesion in chloroquine intoxication. Exp Pathol 1972; 7: 337-341. Matsuzawa Y, Hostetler KY: Effects of chloroquine and 4,4’ (Diethylaminoethoxy) o$Diethyldiphenylethane on the incorporation of [3H] glycerol into the phospholipids of rat liver lysosomes and other subcellular fractions, in vivo. Biochemicia et Biophysics Acta 1980: 620: 592-602. Gonzalez-Noriega A, Grubb JH, Talkad V, Sly WS: Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J Cell Biol 1980; 85: 839-852. Tietze C, Schlesinger P, Stahl P: Chloroquine and ammonium ion inhibit receptor-mediated endocytosis of mannoseglycoconjugates by macrophages: apparent inhibition of receptor recycling. Biochem Biophys Res Commun 1980; 93(i): i-a. Blitz AL, Fine RE, Toselli PA: Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum. J Cell Biol 1977; 75: 135-147. Panayi GS, Neil WA, Duthie JJR, McCormick JN: Action of chloroquine phosphate in rheumatoid arthritis. Ann Rheum Dis 1973: 32: 316-318. Hurvitz D, Hirschhorn K: Suppression of in vitro lymphocyte responses by chloroquine. N Engl J Med 1965; 273(l): 23-26. Norris DA, Weston WL, Sams WM Jr: The effect of immunosuppressive and anti-inflammatory drugs on monocyte function in vitro. J Lab Clin Med 1977; 96(3): 569-586. Ward PA: The ChemOsuppreSSiOn of chemotaxis. J Exo Med 1966; 124: 209-225:. Keen JH, Willingham MC, Pastan IH: Clathrin-coated vesicles: isolation, dissociation and factordependent reassociation of clathrin baskets. Cell 1979; 16: 303-312. Woodward MP, Roth TF: Coated vesicles: characterization,
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selective dissociation, and reassembly. Proc Nat1 Acad Sci USA 1978; 75: 4394-4398. Schook W, Puszkin S, Bloom W, Ores C, and Kochwa S: Mechanochemical properties of brain clathrin: interactions with actin and cu-actininand polymerization into basketlike structures or filaments. Proc Natl Acad Sci USA 1979; 76: 116-120. Allison AC, Mallucci L: Lysosomes in dividing cells, with special reference to lymphocytes. Lancet 1964; II: 1371-1373. Goldstein JL, Brunschede BY, Brown MS: Inhibition of the proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and triton WR 1339. J Biol Chem 1975; 250(19): 7854-7862. Ohkuma S, Poole B: Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 1978; 75(7): 3327-3331. Fedorko ME, Hirsch JG, Cohn ZA: Autophagic vacuoles produced in vitro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine. J Cell Biol 1968; 38: 392-402. O’Brien RL, Allison JL, Hahn FE: Evidence for intercalation of chloroquine into DNA. Biochim Biophys Acta 1966; 129: 622-624. Scholnick PL, Epstein J, Marver HS: The molecular basis of the action of chloroquine in prophyria cutanea tarda. J In-
July 18, 1993
The American Journal of Medicine
58.
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vest Dermatol 1973; 61: 226-232. Neil1 WA, Panayi GS, Duthie JJR, Prescott RJ: Action of chloroquine phosphate in rheumatoid arthritis. II. Chromosome damaging effect. Ann Rheum Dis 1973; 32: 547-550. Michael RO, Williams GM: Chloroquine inhibition of repair of DNA damage induced in mammalian cells by methyl methanesulfonate. Mutat Res 1974; 25: 391-396. Kim SH, Kim JH. Fried J: Enhancement of the radiation response of cultured tumor cells by chloroquine. Cancer 1973; 32: 536-540. Cohen SN, Yielding KL: Further studies on the mechanism of action of chloroquine: inhibition of CNA and RNA polymerase reactions. Arthritis Rheum 1964; 7: 302. Salmeron G, Lipsky PE: Immunosuppressive activity of chloroquine: inhibition of human monocyte function. Arthritis Rheum 1982; 25(suppl): 5132. Elner VM, Schaffner T, Taylor K, Glagov S lmmunophagocytic properties of retinal pigment epithelium cells. Science 1981; 211: 74-76. Bernstein H: Drugs and ocular tissues. In: Dikstein S, ed. Proceedings of the Second Meeting of the International Society for Eye Research. Jerusalem 1976; 575-598. Mackenzie AH, Scherbel AL: Let us abandon some chloroquine dogmas [abstr]. Arthritis Rheum 1969; 12: 315. Popert AJ: Chloroquine: a review. Rheumatol Rehabil 1976; 15: 235-238.
ALLEN H. MACKENZIE, M.D. Cleveland, Ohio
The pharmacokinetics, physiologic effects, and the metabolization of chloroquine and hydroxychloroquine are all similar. Their concentrations in plasma and tissue are directly related to dally dosing. The highest concentrations are found in melanin-containing tissues, particularly the choroid and ciliary body of the eye. The pharmacologic effects of 4-aminoquinoline compounds are reviewed in detail. It is likely that the rheumatologic effectiveness of these agents is primarily related to lysosomal actions. The drug-induced lysosomal abnormalities include diminished vesicle fusion, diminished exocytosis, and reversible “lysosomal storage disease.” It is likely that the retinal toxicity of these drugs is one manifestation of the altered lysosomal physiology involving the retinal pigmented epithelium. Tissue of retinal pigmented epithelium is similar to that of the bone-marrow-derived macrophage. Depression of extra-oculogram is an early sign of excessive dosage and can be used to measure potential toxicity during therapy with 4-aminoquinolines. Dosages ranging from 3.5 to 4.0 mg/kg per day for chloroquine and 6.0 to 6.5 mg/kg per day for hydroxychloroqulne are clinically safe. The beneficial effects of 4-aminoquinoline compounds in the management of rheumatoid arthritis, systemic lupus erythematosus (SLE), and other connective tissue diseases were discovered through serendipity [ 1,2]. While subsequent controlled studies have amply demonstrated the efficacy of these compounds in treating rheumatoid arthritis [3-111, and have established a level of efficacy for the management of SLE, use in juvenile rheumatoid arthritis [2,12] is investigational. They should only be used under approved protocols by experienced clinicians. METABOLISM
From the Department of Rheumatic and Immunologic Disease, Cleveland Clinic Foundation, Cleveland, Ohio. Requests for reprints should be addressed to Dr. Allen H. Mackenzie, Department of Rheumatic and Immunologic Disease, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44106.
Currently, the two most used 4-aminoquinoline compounds are chloroquine phosphate, more popular in Europe [ 13,141, and hydroxychloroquine sulfate, more popular in the United States [ 15-l 7] _ These two agents are very similar antimalarials, differing only in a single hydroxyl group at the end of the side chain. Both are also similar in kinetics [ 181, actions [ 1,191, and metabolism [20,21]. I will therefore treat them alike; what I say of one, largely applies to the other. These basic amines have a heterocyclic planar rl-aminoquinoline nucleus at one end of the polar molecule and a lipophilic side chain on the other end [22], and their kinetics have been fairly well studied [ 181. They are rapidly and completely absorbed when given by mouth [21], and approximately 50 percent are transported by binding to serum proteins [23,24]. Their elimination proceeds in two stages. In the first stage there is rapid excretion with a half-time of
July 18, 1983
The American Journal of Medicine
5
PLAQuENILSYMPOS~UM--MACKENZIE
EXCRETION
INTESTINE
5%
’
and spleen; yet higher concentrations occur in kidney, bone marrow, lungs, and liver, while even higher concentrations are found in the adrenal glands and pituitary and the highest of all occur in melanin-containing tissues, particularly the choroid and ciliary body of the eye [20,28-301. Concentrations several hundred times those in the serum are found in liver, where an approximately 1 X 10e4 M concentration is found at therapeutic serum concentration (1 X lo+ M) [ 19,3 l-331. The therapeutic serum concentrations attainable in man at a clinically safe dosage of chloroquine (3.5 to 4.0 mg/kg per day) [34], produces serum levels of 0.6 to 0.9 pmol/L 119,311. For hydroxychloroquine, safe daily dosage (6 to 6.5 mg/kg per day) 1341 produces serum levels of 1.4 to 1.5 pmol/L [ 19,311. PHARMACOLOGIC
I...
___
Fbure 1.
Diagram of the kinetics, distribution, therapeutic tissue concentration levels, anddisposition of hydroxychloroquine in man 1331.
about three days. The second stage has a more prolonged half-time of about 18 days. Clinical half-life, on the other hand, is 50 to 52 hours for each agent, rising to a higher value at very high serum concentrations [ 181 (Figure 1). About 50 percent of the administered dose has been identifiably recovered [21]. Thus, of the identifiable 4-aminoquinoline compounds excreted, 50 to 60 percent is in the urine, while 8 to 10 percent of chloroquine and up to 15 to 24 percent of hydroxychloroquine is fecally excreted [ 251. Hydroxychloroquine is reportedly conjugated with glucuronide and excreted in the bile [ 20,211. Unknown amounts of 4-aminoquinoline compounds are deposited into dermal cells and appendages, and sloughed off by the skin [ 261. The remainder is thought to be metabolically biotransformed to molecules not readily perceived as being derived from 4-aminoquinoline compounds [ 201. The 4-aminoquinoline compounds attain plasma concentrations that are directly related to the magnitude of the daily dosage [ 231 in a clear-cut, dose-response relationship (Figure 2). At a given serum concentration, the drug is distributed throughout various body tissues, depending upon tissue affinity for the compound [ 271. Fat, bone, tendon, and brain contain relatively small amounts of 4-aminoquinolines [ 201. Lowest tissue concentrations are found in skeletal muscle, skin, and sclera; the next higher concentrations are found in heart
6
July 16, 1963
The Am&can
Journal of Medicine
EFFECTS
Although a broad spectrum of biologic actions of 4aminoquinolines is demonstrable in vitro. or in animals at higher concentrations, these effects are not clinically valuable because they require dosage levels that are too toxic to permit prolonged therapy. Thus, the actions of antimalarial drugs at the above-mentioned.concentrations are the only effects safely attainable in vivo [ 11. The spectrum of pharmacologic actions that pertain in rheumatology probably span the concentration range from 10m7 M to 10m4 M in tissues, but never higher than about 1.5 X 10m6 M in serum. In this concentration range, three principal groups of effects are observable: 1. The 4-aminoquinolines form a complex with ferriprotoporphyrin IX, an intermediary product of hemoglobin digestion by malarial parasites (plasmodia) (351. This complex is so toxic that ions are lost from the parasite cells, osmotic lysis occurs, and malaria is inhibited or cured. Plasmodia resistant to the chloroquine action digest little hemoglobin. Such resistant plasmodia appear no more susceptible to chloroquine than are the cells of the host. 2. Chloroquine influences the behavior of lysosomes, interfering with the vesicle fusion process in a cell [36,37]. Lysosomes increase in number, increase in volume, decrease in density, and develop lamellar membrane structures called myeloid (or myelin) bodies [38-411. Such myelin bodies are thought to be phospholipid configurations [37,41] in phagocytic or autophagic vacuoles. Treated cells are unable to proceed at normal rates with orderly pinocytosis 1421, exoplasmosis, and phagolysosomal fusion [43], with the result that large quantities of cellular membrane are sequestered within the cell in the increased number of lysosomal vesicles. These vesicles contain or consist of plasma membrane phospholipids 1411, with attached cell receptors [ 421. This population of vesicles normally
PLAQUENIL SYMPOSIUM-MACKENZIE
10-5M
500 mg/d
(13 3mg/Kg/d)
10e6M
250mg/d
(4 mg/Kg/d)
/
10e7M 500mghk
Figure 2. A family of curves showing the time course to attainment of plateau concentrations of chloroquine in plasma, and the dependence of plateau plasma concentration on daily dosage rate. While these curves describe the dynamics of chloroquine, hydroxychioroquine behaves in much the same way [d-6,25,26].
(I 2mg/Kg/d (0 6mg/Kg/d
0
eqwalent) equwalent)
2
I MONTHS
I
recycles intact units of membrane rapidly from the surface of the cell into pinocytic and phagosomal vesicles, to the endoplasmic reticulum and golgi, thence back to the cell surface. Primary lysosomes possess a membrane composition similar to that of the plasma membrane [44]. The entire cell surface membrane of a working phagocyte of the macrophage type is recycled approximately every five minutes [42]. A number of specific absorptive endocytosis systems are thought to require this membrane with its receptors in order to operate efficiently. Sequestration of so much plasma membrane in the stabilized vesicles depletes the cell surface of receptors for all manner of molecules, because these receptor sites are imbedded into the phospholipid plasma membrane [42,43]. The depletion of cell surface receptors probably diminishes the rate or efficiency with which a cell responds to its environment. This has been well proven to be true for acid hydrolases [42] of several types, but probably applies also to other receptors, for example, those for phytohemagglutinin. Phytohemagglutinin responsiveness is diminished in chloroquine-treated cells [45,46]. Thymidine uptake has been shown to be diminished. However, the 4-aminoquinoline compounds do not affect receptor affinity [42]. Depletion of cell surface receptors caused by reversible inhibition of membrane recycling appears to be the mechanism, rather than blockade of receptors [43]. Complex functions, such as phagocytosis [47] and chemotaxis [48], are probably dependent on these receptors. Although these effects are completely reversible [42], cellular efficiency is greatly diminished [36]. The growth of cells is inhibited, and a significant amount of cell death will occur at concentrations in excess of therapeutic levels [46]. The mechanism by which the 4-aminoquinoline drugs inhibit vesicle fusion is not yet clear. Some data suggest a chloroquine/clathrin interaction in which the latter is solubilized [49]. Clathrin is a structural protein, coating vesicle walls, which is probably involved in vesicle fusion [44,50,51]. The properties of clathrin are
altered by lysosomotropic amines, elevated pH, and other factors [49]. 3. In the presence of chloroquine or hydroxychloroquine, the digestive efficiency of phagolysosomes is diminished [38,52,53]. Studies have shown delayed digestion during cellular autophagy in the presence of 4-aminoquinoline drugs for several differing constituents: (1) some cellular proteins, (2) mucopolysaccharides, (3) hormones, and (4) phospholipids [53]. The controlled autophagy necessary to the early stages of derepression in a transforming lymphocyte [46,52] or in a cell preparing to undergo mitosis is delayed. The basic amine molecules become concentrated in the lysosomes. The drug passively diffuses into the cell in a lipophilic basic unprotonated state, but once it enters the acid milieu of a lysosome (pH about 4.0), the 4aminoquinoline compound becomes progressively protonated [49]. Protonation renders the side chain more lipophobic, and hence traps the drug within the lysosome. The entry into the cell is by passive diffusion, not requiring energy, but the trapping mechanism depends upon the proton pump in the wall of the lysosome, which does require energy. The pH of a chloroquinetreated lysosome rises from about 4.0 or 4.5 to approximately 6.0 pH units [54]. It is not certain how hydroxychloroquine and chloroquine interfere with lysosomal digestion. Since the pH rises within the lysosome, it conceivably could rise enough to exceed the optimal range for acid hydrolases to perform their digestive functions. In addition, the dissociation of the lysosomal hydrolases from their mannosyl recognition marker “receptor” does not take place with normal speed in the presence of chloroquine [42]. This inhibits the discharge of contained enzymes into a phagosome or to the cell surface. There seems to be a real likelihood that such important cellular activities as feeding, secreting, excreting, synthesis, mitosis, immune transformation, and autophagy [55] depend upon a functional lysosomal system. Thus, the sequestration of receptors within the altered lysosomes could depress immune
July 18, 1883
The American Journal of Medicine
7
PLAQUENIL SYMPOSIUM-MACKENZIE
responsiveness by depletion of appropriate receptors. Add to this the delayed vesicle fusion and inhibited phagosomal digestion, and the outcome is considerable reversible interference in cell work. 4. The 4-aminoquinoline compounds are bound to numerous tissue constituents such as nucleoproteins [ 561, melanins [ 281, porphyrins [35,57], and the like. This begins to be significant at about 1 X 10m6 M serum concentration and becomes much more intense at higher concentrations. The effect on nucleoprotein alters the physical properties of DNA and RNA, thus delaying excision and repair and diminishing the efficiency of polymerases [ 58-611. Effects attainable at still higher concentrations do not appear to be therapeutically significant. In summary, it appears likely that the rheumatologic effectiveness of the 4-aminoquinoline drugs is related primarily to the lysosomal actions. These take place at concentrations safely attainable in the plasma of patients during therapy. ABNORMAL
LYSOSOMAL
FUNCTIONS
Three 4-aminoquinoline-induced abnormalities of lysosomal function are known to occur: 1. Chloroquine diminishes vesicle fusion, perhaps by modifying clathrin [49]. This sequesters large quantities of plasma membrane in the lysosomes, thus trapping as much as 50 percent of receptors within, away from the cell surface [42]. This would appear to alter the cell’s responsiveness, not only to many ordinary stimuli, but probably also to immune stimuli. 2. Exocytosis of cell products is diminished by the same inhibition of vesicle fusion. Salmeron and Lipsky [62] have found lnterleukin 1 concentration to be greatly diminished in vitro when using very low concentrations of chloroquine. Conceivably, lnterleukin 1 is secreted via exocytosis of transport vesicles, via carrier-receptors, or via both. Cells rich in lysosomes appear particularly susceptible to these effects of the 4-aminoquinoline compounds. Polymorphonuclear leucocytes, macrophages, synovial macrophages, and retinal pigmented epithelium macrophages are all altered in morphology and in biologic responses by 4aminoquinolines in therapeutic concentrations. 3. Lie and Schofield [38] have drawn the analogy of resemblance to a “lysosomal storage disease” during therapy with chloroquine. Storage results when diges-
tive processes cannot proceed to completion, so that residual matter is left in lysosomes/phagosomes unable to be exocytosed. RETINAL
PIGMENTED
EPITHELIUM
Elner et al [63] have put forth evidence that the retinal pigmented epithelium is a tissue very similar in its structure and responses to bone marrow-derived macrophages. The cells of retinal pigmented epithelium possess microvilli and numerous ruffles under electron microscopy, and they demonstrate phagocytosis, glass adherence, and the ability to phagocytose erythrocytes coated with antibodies. It is the duty of the retinal pigmented epithelium to scavenge, to phagocytose, and intracellularly to digest the aged photoreceptor membrane discs in its phagolysosomes. Photoreceptor discs are shed diurnally from the tip of the rod and cone cells under the influence of light. Potts [29] has demonstrated dose-related depression of retinal pigmented epithelium metabolism, which is reversible up to very high drug concentration levels. The photoreceptor lesion appears to be largely secondary to retinal pigmented epithelium depression during 4-aminoquinoline therapy, and it too has been reversible up to the point of extensive cell death, which correlates with excessive daily doses. The biologic current generated in the retinal pigmented epithelium is measured as the extra-oculogram. Depression of the extra-oculogram is an early sign of excessive 4-aminoquinoline dosage, and has been used to monitor eyes during therapy [ 141. It is reasonable to suspect that a dosage-dependent, pharmacologicinduced lysosomal storage disease of the retinal pigmented epithelium might cause retinal dystrophy from inefficient elimination of photoreceptor cell debris. It is also reasonable to suspect that a threshold level of this phenomenon can be defined in terms of dosage or concentration, above which toxicity develops but below which therapy may be conducted in relative safety [34,64,65]. Since the retinal lesion is the only medically significant toxicity of antimalarial drugs [66], all other adverse effects being reversible, studies were undertaken to attempt to define a therapeutically safe zone, and the threshold of toxicity. These will be discussed in my paper, “Dose Refinements in Long-Term Therapy of Rheumatoid Arthritis with Antimalarial%” appearing elsewhere in this symposium issue.
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The American Journal of Medicine
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