DRUG DISPOSITION

Clin. Pharmacokinet. 21 (4): 274-284, 1991 0312-5963/91/00 I0-0274/$05.50/0 © Adis International Limited. All rights reserved. CPK1066

Interleukins Clinical Pharmacokinetics and Practical Implications Velio Bocci Institute of General Physiology and Nutritional Sciences, Faculty of Pharmacy, University of Siena, Siena, Italy

Contents 274 275 276 279 279 280 280 280 280 281 28 J

Summary

Summary I. Pharmacokinetics of Interleukins 2. Efficient Catabolism May Prevent Toxicity 3. Toxicity of Interleukins and TNF 4. The Search for Optimal Delivery Systems 4.1 Oral and Rectal Administration 4.2 Administration by Inhalation 4.3 Transdermal Delivery 4.4 Conventional Delivery 4.5 Delivery Via the Lymphatic System 5. Conclusions

Interleukins and tumour necrosis factor (TNF) are a complex group of proteins and glycoproteins able to exert pleiotropic effects with respect to a number of different target cells. In physiological conditions, they are induced and released in basal amounts only in restricted microenvironments where they have paracrine activity. Any small amounts reaching the circulation do not disturb homoeostasis. During therapy, particularly when these cytokines are administered via conventional routes, it has become apparent that their presence in non physiological plasma concentrations and their unselective action cause toxic effects with small benefits. The pharmacokinetics of interleukins-l, -2, -3 and -6 and TNF have been evaluated, and their disappearance from plasma after intravenous administration is very rapid (i.e. the distribution half-life is measured in minutes; the elimination half-life is several hours). The efficiency of catabolic pathways such as renal filtration and/or liver uptake is interpreted as a salutary mechanism for extracting proteins that should not be in the circulation. However, because these cytokines are very potent immunomodulatory agents there is a need to improve their therapeutic index, and to this end a number of possible formulations and routes of administration are now available and may eventually be of practical use.

Interleukins are a family of proteins and glycoproteins produced by several cell types (T lympho-

cytes, macrophages, endothelial cells and fibroblasts) mostly implicated in the control of immune

275

Clinical Pharmacokinetics of Interleukins

reactions, haemopoiesis and lymphopoiesis. To date as many as I I interleukins have been identified and cloned but, taking into account the interferons (a, /3 and ")'), tumour necrosis factor (TNF-a and -/3), colony-stimulating factors (CSF) and erythropoietin, it becomes clear that these cell products (or cytokines) represent the first basic words of what is probably a more complex cell language. For brevity this article does not attempt to review all the available data on the molecular biology, biochemistry and immunology of cytokines, and it suffices to note that their molecular weights range from 14 to 22kD, although TNF-a is a trimeric molecule (Jones et al. 1989); the isoelectric points vary from pH 5 to 7 and, with a few exceptions such as human TNF-a (Marmenout et al. 1985), these molecules contain natural polypeptide chains with glycosylation sites for the attachment of oligosaccharide chains (Conradt et al. 1985). At least in vitro, the latter are not necessary for expressing biological activity (Rosenberg et al. 1984) but glycoproteins in vivo, in comparison with recombinant unglycosylated proteins, appear to be more stable and probably less immunogenic, and to have a slightly prolonged half-life in the circulation, the typical case being that of erythropoietin which, unless g1ycosylated, is therapeutically inactive (Bocci 1983; Siegel et al. 1985). So far, however, these somewhat controversial advantages have been overridden by the difficulty and cost of producing large quantities of natural cytokines, and only the cloning of human genes in eukaryotic cells can allow production of large amounts of material for human use. Most of the target cells have high affinity receptors (from about 100 up to 50 000 per cell) for all of these factors and this explains why cytokines when used pharmacologically display pleiotropic effects, not all of them desirable. Moreover, while cytokines in physiological conditions are essential for the correct functioning of the immune system, in certain diseases they have important pathological implications (Hirano & Kishimoto 1989; Metcalf 1990). The purpose of this review is to examine the pharmacokinetics of some cytokines in laboratory

animals and, whenever possible, in humans. Attention is focused on the distribution and metabolism of interleukin (IL)-I, IL-2, IL-3, IL-6 and TNF-a, since pharmacokinetic data are not yet available for the remaining interleukins. The metabolism of interferons has been reviewed elsewhere (Bocci 1987, I 990a). Even with these limitations it is already possible to get a fair understanding of the catabolic system which efficiently removes interleukins from the circulation. Finally, in view ofthe practical applications in malignancies, immunodeficiencies, autoimmune diseases and infectious diseases, it appears to be relevant to evaluate future avenues of research that may either improve the therapeutic index of these natural drugs, or counteract their detrimental effects.

1. Pharmacokinetics of Interleukins An interesting feature of interleukins is their middle-range molecular weight, in that it is not as small as that of insulin nor as large as that of albumin. It would seem that during the course of evolution the size, shape and electric charge of these ligands have adapted to the necessity of a paracrine mode of action, interacting with several types of receptors and large numbers of cells (Bocci 1985b). From a strict physiological point of view, the analysis oftheir pharmacokinetic properties would be unnecessary because interleukins are normally produced and released in microenvironments in which they both act and are catabolised (Bocci 1988a); any trace which spills over and reaches the circulation is necessarily diluted and eliminated so rapidly as to become undetectable in the peripheral plasma. However, as these compounds are now used as drugs it becomes necessary to know their patterns of distribution and elimination. Because their disappearance from plasma is generally so rapid, it has not been easy to carry out detailed and precise pharmacokinetic studies. Moreover, while both the immunolabelled and radioiabelled interleukins can be measured accurately in plasma, these values do not always reflect the movement of the endogenous protein which, when measured by cur-

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rent bioassays, presents a certain degree of inaccuracy. Despite these difficulties and on the basis of previous studies on interferons (Bocci 1985c, 1987) it is possible to draw a definitive picture: when interleukins are injected into the plasma pool, they mix rapidly within about 2 or 5 min, in mice and humans, respectively, depending on the heart rate. Importantly, the assumption that the first sample taken after that time represents the total of the dose mixed in the plasma volume may be incorrect, because even during the first few minutes, up to half of the dose could be taken up by cells and catabolised. Thus, the correct procedure would be to measure the plasma volume independently and calculate the theoretical interleukin concentration at the time of administration so that all the following experimental points can be referred to as a percentage of the first value. When this was done, we found that the plasma half-life (t,/,) of the distribution phase was considerably shorter than that currently calculated (Bocci et al. 1985b). Another problem arises with the difficulty of measuring plasma concentrations for a time long enough to precisely ascertain the final exponential slope. There is little doubt that the real disappearance curve is multiexponential, i.e. described by at least the a , f3 and "y phases , (Bocci et al. 1985b). This would depict realistically the complexity of the distribution of cytokines into compartments with different permeability and hence with dissimilar transfer rates. However, for the sake of simplicity, a semilogarithmic plot is usually made of interleukin concentration in the central compartment (plasma) versus time, thus describing both distribution (t,/,u) and elimination or metabolic (t'/'/l) phases with only a 2-compartment open model (Greenblatt & Koch-Weser 1975). This appears to be sufficient in most cases because, in practical terms, the activity of biological response modifiers depends more on the prolonged cellular response than on a constant plasma concentration. Table I contains available values for interleukins and TNF-a. The considerable variability reported by different researchers supports the contention that values cannot be considered absolutely

c/in. Pharmacokinet. 21 (4) 1991

precise. However, they clearly indicate that interleukins have an extremely rapid elimination from the plasma after intravenous bolus injection. Prolonged half-lives (of several hours) have been noted after intraperitoneal, intramuscular and subcutaneous administration: as an example, IL-2 remains detectable in the plasma for about 8h after intramuscular and subcutaneous administration, and a bioavailability of about 30% has been calculated for these routes (Cheever et al. 1985; Konrad et al. 1990). While IL-6 was already absent from plasma 2h after intravenous administration, it persisted for more than 6h after an intraperitoneal dose (t,/, = 3h) although, in the latter case, the dose was 10 times higher (Mule et al. 1990). When escalating doses of either IL-2 or TNF-a have been administered intravenously to animals and patients, a progressive prolongation in half-lives and a reduction in the apparent volume of distribution (Vd) have been noted, indicating the relevance of cell receptor binding at very low doses (Blick et al. 1987; Kimura et al. 1987; Konrad et al. 1990) and a significant prolongation of the t'/'/l' particularly in female rats (Ferraiolo et al. 1989). An unusual finding was the detection (by both radioactive and biological assays) ofTNF-a in rabbit plasma after oral administration, suggesting the possibility of a small but effective absorption process via the oropharyngeal mucosa (Pacini et al. 1987).

2. Efficient Catabolism May Prevent Toxicity The question whether the efficient catabolism of interleukins may prevent toxicity has previously been discussed at length (Bocci 1987; 1990a). The rapid disappearance of interleukins from plasma can be summarised briefly here. First, interleukins are diluted in the plasma and interstitial pools, and it is possible that they undergo inactivation in blood by proteinases (Konrad et al. 1990), or by inhibitors such as a soluble TNFreceptor (Engelmann et al. 1989; Seckinger et al. 1990), or such as a group of molecules inhibiting IL-I (as reviewed by Larrick 1989), IL-2 (Kucharz

Clinical Pharmacokinetics of Interleukins

277

Table I. Pharmacokinetic parameters of interleukins and tumour necrosis factor Protein

Tested in

t'h6 (min)

tV2a

(min) Rabbit leucocyte pyrogen Rat IL-1 Human r IL-1 jS Human rlL-1jS Human rlL-1 jS Murine IL-2 Murine IL-2 Human IL-2 Human r1L-2 Human IL-2 Human r1L-2 Human r1L-2 Human IL-2 Human r1L-2 Human r 125_IL_2 Human r1L-2 Murine IL-3 Human r 1251_IL_3 Human r unlabelled IL-3 (bioassay) Human r 1251_IL_6 Human r1L-6 1251_TNF Murine TNF RatTNF Human rTNF Human rTNF Human rTNF Human rTNF Human rTNF (1-16 x 105 U/m2) Human rTNF Human rTNF Human rTNF: (10-46 ltg/kg) (10-63 ltg/kg) Human rTNF (0.04-0.16 mg/m2) (0.20-0.28 mg/m2)

Abbreviations: tv...

Rats Rats Mice Rats Rats Mice Mice Humans Mice Humans Humans Mice Humans Humans Mice Humans Mice Mice Mice Rats Mice Mice Mice Rats Mice Humans Rabbits Monkeys Humans Humans Humans Mice Rats d Rats 9 Humans

6-10 ",2 ",27

"'5 "'3 3.36

"'240 19

"'3 3.7 ± 0.8 "'22.5 1.6 ± 0.3 30-120

"'6 6.9 7-9 7-10 7-10 5.7 12.9

"'60 > 90

Kampschmidt & Upchurch (1980) Kampschmidt & Jones (1985) Newton et al. (988) Klapproth et al. (1989) Kudo et al. (1990) Muhlradt & Opitz (1982) Donohue & Rosenberg (1983) Bindon et al. (1983) Chang et al. (1984) Lotze et al. (1984) Lotze et al . (1985) Cheever et al. (1985) Siegel et al. (1985)

"'90 85

< 30 3-5

300 50

"'30 3

"'55

"'7 6-7 10.5 27 ± 7 32 14-18 32-51 80

Ohnishi et al. (1990) Konrad et al. (1990) Garland et al. (1983) Metcalf & Nicola (1988) Castell et al. (1988) Mule et al. (1990) Beutler et al. (1985) Flick & Gifford (1986) Waage (1987) Asher et al. (1987) Blick et al. (1987) Bocci et al. (1987a) Creaven et al. (1987) Kimura et al. (1987)

"'25 10-80 ",24 18.5-19.2 2.1 2.6

Reference

102-162 14.4-31 .8 18.0-35.3

Feinberg et al. (1988) Ferraiolo et al. (1988) Ferraiolo et al. (1989) Moritz et al. (1989)

11 -17 54-71

= distribution half-life; "h~ = elimination half-life; IL = interleukin; r = recombinant; TNF = tumour necrosis factor.

& Goodwin 1988) and interferon (Fountoulakis et al. 1990; Jiang et al. 1983; Lefkowitz & Aeischmann 1985; Novick et al. 1989). They might also be inactivated by binding to carriers such as a2macroglobulin (Borth & Luger 1989; James 1990; Matsuda et al. 1989) or autoantibodies (Bendtzen

et al. 1990; Bocci 1991). The relative extent of these processes has not yet been determined in physiological conditions. Secondly, interleukins bind to high affinity functional receptors within cell membranes. Unfortunately they are not selective ligands and when

278

they are present in the blood stream, it is probable that only a small amount becomes bound to appropriate immunocompetent cells, while the bulk interacts with endothelial and parenchymal cells. Interleukins can bind to glial, neurosecretory and neuronal cells of the hypothalamus via the permeable capillaries of the circumventricular organs, thus explaining the variety of observed CNS side effects and hormonal changes associated with these agents (Blatteis 1990; Bocci 1988b; Krueger et al. 1990; Shibata 1990; Stitt 1990). Thirdly, interleukins are eliminated mainly by the kidney, and possibly the liver, while the gut, lung and other organs usually playa negligible role. The importance of the kidney in this respect was first hypothesised in 1977 (Bocci) and later proved for interferon (lFN)-a and -{3 (Bocci et al. 1981, 1982). Moreover, it has been shown that the bulk of circulating IL-I (Klapproth et al. 1989; Kudo et al. 1990; Newton et al. 1988; Townsend & Cranston 1979), IL-2 (Donohue & Rosenberg 1983; Konrad et al. 1990), IL-3 (Metcalf & Nicola 1988) and TNF-a (Ferraiolo et al. 1989; Pessina et al. 1987) is eliminated by the kidney. Renal catabolism occurs in the following steps: filtration by the glomerular sieve, reabsorption of interleukins by the proximal tubules possibly accompanied by peri tubular uptake, degradation in the tubular cells and finally 'elimination of proteolytic fragments. No measurement is available of the glomerular sieving coefficient (GSC) [expressed as the ratio of interleukin clearance to creatinine clearance (CLcR)] but, as interferons are similar proteins to interleukins, it is probably in the range of 50 to 60% depending on the size, shape and charge of the interleukins. If this is correct, assuming a renal plasma volume of about 650ml and a glomerular filtration rate (GFR) of about 120 mlf min it will mean that the human kidney can clear the whole plasma volume of interleukins in 40 to 50 minutes (Bocci 1987). This time would be considerably shortened if the GSC is higher than 60% and if there is a concomitant peri tubular uptake, which is very likely because tubular cells may have receptors for some interleukins on the basolateral membrane.

C/in. Pharmacokinet. 21 (4) 1991

When tubular cells are normal, they can reabsorb interleukins almost entirely quantitatively and this seems to occur at least for IL-2 (Donohue & Rosenberg 1983) and IL-3 (Metcalf & Nicola 1988). IL-l present in the tubular fluid may either undergo a partial hydrolysis at the brush border - this would explain the finding of IL-I fragments in human urine (Kimball et al. 1984) - and/or may be directly absorbed. An alternative explanation could be that the human kidney has less reabsorptive capacity than rat kidney (Kudo et al. 1990). Once interleukins or albumin have been internalised in tubular cells, hydrolysis occurs in the lysosomes mainly by the action of cathepsin D (Baricos et al. 1987; Ohnishi et al. 1990) and it is generally agreed that intact proteins are not returned into the peritubular fluid (and hence to the circulation). An attempt to inhibit degradation of IL-l (Kudo et al. 1990) and IL-2 (Ohnishi et al. 1990) in the Iysosomes by administration of several inhibitors in vivo has revealed that pepstatin A, an inhibitor of aspartic proteinases, is most effective. An intriguing result that deserves further investigation is that pepstatin A appears to prolong the plasma t,;, of both IL-I and IL-2 but the mechanisms of this delay are unclear, as it might be due to either diminished filtration for partial inhibition of the renin-angiotensinogen system and/or blocked protein catabolism in the lysosomes with exocytosis of lymphokines into the peri tubular fluid and thence into the circulation. This latter possibility deserves further investigation because it could clarify whether, if proteolysis cannot proceed, proteins could undergo vesicular transport (Milici et al. 1987) from the luminal to the basolateral pole without fusing with lysosomes also in the kidney. It may be useful to add a brief comment regarding pathological changes of the kidney: on the one hand, any reduction of the GFR is likely to prolong the half-life of interleukins in the circulation although, in chronic glomerular damage, proteins may abnormally leak out from the peritoneal and/or gut surface. On the other hand, tubular damage implies a diminished absorption of proteins with increased loss of intact or partly degraded interleukins in the urine.

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Clinical Pharmacokinetics of Interleukins

Fourthly, the liver can be another important site of interleukin catabolism by virtue of either functional and/or clearance receptors, i.e. hepatic lectins (Ashwell & Harford 1982). Both (probably in different amounts) are located in hepatocytes, Kupffer cells and endothelial cells, and lead to an array of biological effects and/or catabolism. Most natural interleukins are glycoproteins with different terminal sugar signals such as galactose, glucosamine and fucose, recognisable by several hepatic binding proteins, but it appears that unglycosylated proteins (such as recombinant IFN(3 and -I') are also rapidly taken up by as yet unidentified cell receptors (Bocci et al. 1985a, 1987b). Thus, it is not possible to define in quantitative terms the catabolic role of the liver for interleukins except that up to about 80% of the recombinant human 125I-IL-6 was bound to hepatocytes in a few minutes but (surprisingly) was later released and apparently degraded in the skin (Castell et al. 1989). Recombinant human TNF appears to accumulate more in the liver than in the kidneys of mice (Ferraiolo et al. 1988). Finally, the catabolic role of the gut has not been investigated but is probably marginal unless either the intestinal lining degenerates (after irradiation, chemotherapy, autoimmune diseases, etc.) or portal hypertension develops.

3. Toxicity of Interleukins and TNF An array of toxic effects, somewhat similar to those noted during the early phase of septic shock, have been observed in animals and patients after administration of IL-2 (Gaynor et al. 1988; Klausner et al. 1990; Lee et al. 1989; Lotze et al. 1984) and TNF (Creaven et al. 1987; Feinberg et al. 1988; Gresser et al. 1987; Kimura et al. 1987; Moritz et al. 1989; Spriggs et al. 1988; Stephens et al. 1988). They are due to unphysiologically high concentrations of these cytokines in the circulation and with some approximation they are dose dependent, so that the higher the concentration, the worse is the toxicity. This is not surprising because these cytokines are not supposed to be circulatory proteins (Bocci 1985b, 1988a). Some toxic effects are more

characteristic for I cytokine than another, and in fact IL-2 causes the capillary leak syndrome with cardiorespiratory effects while TNF may instead favour a disseminated intravascular coagulation (DIC) syndrome and metabolic alterations. However, near or at the maximal tolerated dose, there is considerable overlapping of effects such as rigor, chills, fever, hypotension, fatigue, headache, nausea and various mental disabilities up to cerebral coma, mostly because the administration of I cytokine can induce the release of others, i.e. IL-2 causes the release of IFN--y and TNF while the latter causes the release of IL-I and IL-6. With cessation of therapy, side effects disappear more or less rapidly and can be in part relieved by pretreatment with paracetamol (acetaminophen), indomethacin and dopamine hydrochloride. Steroids were effective in reducing weight gain but abrogated the therapeutic effect of IL-2 (Papa et al. 1986). The relationship between cytokines on the one hand and CNS areas, functional changes and symptoms has been discussed in detail elsewhere (Bocci 1988b). Treatment with IL-2 and TNF, particularly after intravenous administration, is followed by a significant increase in plasma cortisol but, surprisingly, this fact, except for interferon (Bocci 1985a), has not been considered relevant for possible immunosuppressive effects.

4. The Search for Optimal Systems

1>elive~

It is now clear that the pleiotropic and redundant activities of interleukins depend on their distribution, their concentration in body fluids and the presence of receptors in target cells. It is important to appreciate that these parameters are deeply influenced either by physiological condition or by acute reactions (such as toxaemia) or pharmacological intervention. In healthy conditions, after induction by trace amounts of lipopolysaccharides bound to several carriers, interleukins are released in highly restricted sites and have paracrine or autocrine actions on neighbouring cells

280

(Bocci 1985b, I 988a). Because most of the interleukins are used within the pericellular site, only small amounts reach the plasma pool via the lymphatic and venous circulation; because of dilution and rapid elimination, their levels become barely detectable and do not alter homeostasis. In contrast, during sepsis, the physiological distribution is subverted because the basal production scales up to a macroscopic process, with interleukins having primarily endocrine effects on a large number of cells which then become responsible for deranged metabolism and toxicity. It has now been realised that interleukins are fairly selective insofar as their normal distribution is restricted to microenvironments while, when present in the circulation, they bind unselectively to endothelial and parenchymal cells and elicit undesired reactions. During the last 10 years, with rare exceptions, interferons and interleukins have been administered to patients with the same pharmacological approach used for antibiotics and cytostatic agents, in the belief that 'the more the better' and disregarding the fact that their mechanisms of both action and distribution are quite different. Only recently has the idea that low doses may be immunologically more active than high and toxic ones begun to emerge and it is to be hoped that during the next few years we will be able to employ interleukins on a rational basis. The following approaches should be evaluated, particularly when interleukins are intended to be used as immunomodulators.

Clin. Pharmacokinet. 21 (4) 1991

and of protein absorption. These processes can be accomplished via the portal-caval blood and, most importantly, via intestinal lymphatics with activation of effector cells present in the liver, lymph and lymph nodes. Obviously, the success of orogastrointestinal and rectal routes depends entirely on the ability to devise delivery systems and pharmaceutical formulations which can inhibit proteolysis and overcome the formidable barrier posed by the gut to the entry of intact proteins. 4.2 Administration by Inhalation In this case interleukins can interact with the rhinopharyngeal and bronchial-associated lymphoid tissues. From these sites immunocytes can transfer and amplify the activation in the spleen, bone marrow and lymph nodes. Interestingly, it has already been shown that only a small amount of interferon delivered via the bronchial route reaches the general circulation in active form, thus minimising side effects (Bocci et al. 1985). 4.3 Transdermal Delivery In comparison with the bronchial-alveolar area the skin surface is small, but it represents an untapped reservoir of resting lymphoid tissue. Important technical developments such as iontophoresis of small proteins have been made in the past few years, but problems still remain to be solved for achieving the passage of proteins through the stratum corneum (Langer 1990; Sanders 1990).

4.1 Oral and Rectal Administration 4.4 Conventional Delivery The oropharyngeal-associated lymphoid tissue is an obvious target for interacting with interleukins with successive amplification of effects within the internal immune system. Experimental results indicate that extremely low doses of interferon or of IL-I, TNF and interferon in breast colostrum can elicit an important priming and stimulatory effect in immunodeficient patients and neonates. Both the vast jejunal and rectal surfaces may become important as sites of both interleukin interaction with the gut-associated lymphoid tissue

The obvious way, but not necessarily the best, is the administration of interleukins by parenteral delivery through the intravenous (bolus and infusion), intramuscular or subcutaneous routes. Although IL-2 has mostly been administered intravenously (Rosenberg et al. 1987; West et al. 1987), this may not be the optimal approach because it subverts the physiological distribution and causes considerable toxicity. Proponents of this route have suggested that alterations of vascular permeability

Clinical Pharmacokinetics of Interleukins

may be important in the development of the therapeutic response (Lee et al. 1989) and that this is the best way to achieve high plasma concentrations of the drug in lymphoid and haematopoietic organs. However, this objective can also be obtained after subcutaneous administration, sparing considerable side effects to the patient and probably achieving similar therapeutic results. Moreover, as noted in section 2, the intravenous route implies an enormous loss of the drug via renal filtration or other catabolic pathways. Administration of interleukins in physiological solution via the subcutaneous route is probably one of the most effective and practical approaches, even though it is unable to abolish side effects, particularly when bioavailability is almost complete and plasma drug concentrations are sustained for a long time. Several biodegradable- and nonbiodegradable-release delivery systems are under development (Langer 1990; Sanders 1990) with the aim of allowing a continuous or controlled release of protein from the site of implantation, and preclinical studies can be expected to be published in the next few years. 4.5 Delivery Via the Lymphatic System Another reasonable approach appears to be the intralymphatic administration of interleukins (Shau et al. 1990) but the necessity of an adequate lymphatic access and of continuous infusion makes it cumbersome and not radically different from a very slow intravenous infusion. An alternative and far simpler approach is the subcutaneous administration of IL-2 in a solution with a very high albumin concentration (20 to 25%) so that the increase of interstitial fluid pressure favours absorption of the interleukins via the lymphatics (Bocci 1984). If administration is carried out in several sites of the subcutis, different areas of the lymphatic system interact with the drug that eventually reaches the plasma pool and hence the spleen and bone marro.w, thus generalising the immunostimulatory effects (Bocci et aI., unpublished data). One problem that has been noted after intralymphatic and subcutaneous administration is the high incidence of

281

antibody formation (Sarna et al. 1990; Whitehead et al. 1990) which could perhaps be alleviated by use of recombinant glycosylated IL-2, which is identical to the endogenous protein. However, albumin may prevent aggregation of recombinant unglycosylated IL-2 at neutral pH, since the present authors have noted an increased bioavailability. Finally, it will be interesting to evaluate the behaviour of polyethylene glycol-modified IL-2 in patients, since in preclinical studies this conjugate appears to have a low clearance, increased antitumour activity and decreased immunogenicity (Katre 1990; Knauf et al. 1988; Zimmerman et al. 1989).

5. Conclusions To date, of the 20 or so available cytokines only interferons, IL-2 and TNF have been extensively tested in patients. Despite serious toxicity, clinical efficacy has been almost nil after administration of TNF except when it was administered intratumourally to prove the point that, if it were possible to achieve very high plasma concentrations (fatal to the patient) of this drug, tumour lysis could be achieved in all sites. Regarding IL-2, clinical responses have been modest and the situation is now at a point where it is necessary to revise at least the routes and modality of administration. Moreover, complex procedures, even if they prove that immunotherapy can be effective in human cancer, are not useful to the millions of patients with neoplasia who could take advantage of self-administration of a drug if not to cure, at least to stabilise the disease. In the preceding section, it is noted that, in comparison with conventional routes of administration, there are now several options that should become areas for intensive research with the aim of improving the therapeutic index. Two further areas deserve investigation: the first concerns the formation of anti-interleukin antibodies which may abrogate the therapeutic efficacy during prolonged treatment. Antibody formation appears to be influenced by immunological status, overall dosage, route of administration and the structure

282

and composition of the recombinant protein. Ifwe can learn the lesson from the interferon field where it has now been clearly shown that natural (endogenous) interferons are far less immunogenic than recombinant ones (Bocci 1991), we ought to switch the production of recombinant interleukins from bacteria to eukaryotic cells as soon as possible. Secondly, there is intense research in the field of interleukin inhibitors and antagonists (Engelmann et al. 1989; Fountoulakis et al. 1990; Larrick 1989; Seckinger et al. 1990), as these may become more useful than antibodies (Tracey et al. 1987) in the treatment of diseases associated with the excessive production of interleukins, which reminds us that these natural proteins can be both friends and foes.

Acknowledgements Thanks go to my wife Helen for revising my English, and to Miss P. Marrocchesi for preparing the manuscript. This work was supported by Contract No. 91.00089.PFAI of the National Research Council (CNR) Targeted Project 'Prevention and control of disease factors' Subproject 02.

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Correspondence and reprints: Professor Velio Bocci, Istituto di Fisiologia Generale, via Laterina 8, 53100 Siena, Italy.