FR2703251A1 - Compositions for application in human therapeutics, characterised by the combination of a muramylpeptide with a cytokine - Google Patents

Abstract

The invention relates to a composition combining, for the purposes of their application in human therapeutics, a natural or recombinant, preferably human, cytokine with a muramylpeptide. It can be used for antiviral or antitumour therapies and/or for promoting reconstitution of the haematopoietic system, especially in people whose immune system is or has been rendered deficient.

Description

COMPOSITIONS FOR THE APPLICATION
IN HUMAN THERAPEUTICS, CHARACTERIZED BY
THE ASSOCIATION OF A MURKMYL-PEPTIDE WITH A CYTOKINE
Interferons (more particularly interferon a) are secreted by leukocytes. In this respect, they are classified as cytokines, mediators produced mainly by the cells of the immune system. In general, cytokines can be considered as characteristic hormones of the immune system. The ability to produce cytokines by genetic recombination has opened the door to more sophisticated studies of the behavior of some of these cytokines, including recombinant interferons in animals. Among the interferons, the most studied is interferon a (IFN a) which is subdivided into IFN a 2a,
IFN a 2b and IFN 2c.

In particular, the antiviral activity of certain mouse interferons, and even the potentiation of this activity in mice by certain N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) derivatives or homologues of those have already been highlighted. On the other hand, as F. Dianzani points out in his article "Interferon Treatments: How to Use an Endogenous System as a Therapeutic Agent" (Interferon Treatments: How to Use an Endogenous System as a Therapeutic Agent) in the "Journal of Interferon Research, Special Issue, May 1992, Mary Ann Liebert, Inc., Publishers, pp.109-118, the numerous clinical trials attempted in humans with interferons have been" more than disappointing ". Although animal tests appeared to open up wide therapeutic potential, their poorly controlled side effects have so far only permitted their use in a limited number of therapeutic indications, so it has been proposed to date. to use IFN at 2a only in a small number of "niches" as also indicated by the "Scrip's Cytokines Report", PJB Publications Ltd. 1993, about the difficulties encountered in the application in human therapeutics of interferons
(a) in the treatment of hepatitis B and C cases,
more serious. Treatments consist of
daily administrations or at the rate of three
once a week from 5 to 10 million units
for 3 to 6 months. Positive results are
observed in 30 to 40% of cases
(b) in the field of cancer, the treatment of
hairy cell carcinoma (Hairy Cell Leukemia) and
chronic myelogenous leukemia,
envisaged applications are AIDS, the syndrome
of Kaposi, multiple myeloma, melanoma and
certain types of carcinoma.

The severity of these syndromes is such that it has also led to the acceptance of the serious side effects accompanying the use of IFN a in human therapy. Ninety-eight percent of the patients treated in this way suffer from an influenza-like illness, which is often extremely severe, accompanied by nausea, vomiting, central and peripheral nervous system disorders and cardiac disorders. These effects are attributed at least in part to the induction by interferon of pyreuric mediators that could be IL-1 and prostaglandins whose production would be activated by interferon. In addition, fatigue, anorexia, weight loss, which may be related at least in part to the production of TNF and / or the increased expression of TNF receptors also induced by interferons. a procedure that would optimize the beneficial effects of therapeutically useful interferons or cytokines while also optimizing the production or activity of cytokines or other undesirable biological mediators such as IL-1, IL-8 and / or TNF (abbreviation for English expression "Tumor Necrosis
Factor "or factor of tumor necrosis), could only be doomed to a certain failure.

 Finally interferon administrations are often accompanied by strong leukopenia and thrombocytopenia accompanied by blocking the maturation of myeloid precursors. These phenomena lead to the need to periodically interrupt the interferon-based treatments, each time to allow a regeneration of the blood count.

 So many disadvantages that, until now, do not allow an exploration of the real possibilities of using interferons in human therapy, except for the treatment of the few syndromes mentioned above.

Similar difficulties are encountered with other cytokines whose therapeutic value would be certain if it were possible to remedy these difficulties. This would be the case, for example, with IL-2,
IL-3, IL-6, G-CSF, M-CSF, GM-CSF, etc., of TNF and IL-1, which themselves have therapeutic potentialities. Their association with muramylpeptides may allow to take advantage of their beneficial effects since they could then be used in non-toxic doses.

It is known that it has been suggested to use in particular IL-3 and certain CSFs to accelerate haematopoietic recovery in patients with marrow aplasia (which is manifested by an endogenous pathogenic factor and / or iatrogenic), especially after chemotherapy or in patients who have undergone bone marrow transplants. But the use of IL-3 in the human clinic or the most active CSFs (eg GM
CSF) encounters in practice the same difficulties as interferon a.

 To these therapeutic disadvantages, are added the extreme costs of current treatments based on therapeutically useful cytokines. The doses administered, in so far as the effect can be regulated in vivo, are indeed considerable compared with the therapeutically useful doses, which can be explained by the fact that unlike the endocrine hormones, the cytokines do not act not at a distance but only on the cells close to the secretory cells. In other words, any cytokine administered that does not reach the target cells can be considered as 1, therapeutically lost.

 The object of the invention is to remedy at least a large part of these disadvantages and difficulties.

 The possibility of benefiting from the therapeutic effects of IFNα or therapeutically useful cytokines other than IFN, where appropriate in smaller doses, without causing unbearable side effects would be a very important step both therapeutically and therapeutically. on the economic plan. This is one of the objectives that the invention has set itself.

 More specifically, the object of the invention is to increase the effectiveness of therapeutics using an interferon or, more generally, a cytokine having a therapeutic value and even to broaden the therapeutic fields of these cytokines (which naturally include interferon), while largely remedying the disadvantages indicated above, more particularly in relation to interferons.

 The invention is largely based on the discovery that human clinically associated administrations of cytokines, for example interferon, and muramyl peptides also induce the synthesis of other cytokines that do not appear, all at least at detectable levels, following the administration of only one of the two elements, interferon and muramylpeptide, and therefore expand the therapeutic activity spectra of cytokines and muramyl peptides. Among the cytokines induced, inter alia, interleukin 6 (IL-6), G-CSF and an antagonist (IL-1 RA) of the interleukin-I receptor. of certain muramyl peptides and interferon a (IFN a) allow the production of the desired effects by using suboptimal doses of cytokine, in particular IFN a, by potentiating the beneficial effects of the cytokine, but in the absence of induction of undesirable cytokines, in particular IL-1, TNF or IL-8.

The invention is not limited to the combination of an interferon (a, ss or a) with a muramyl-peptide. The interferons may also be replaced by other cytokines, particularly those identified above by way of example. In other words, it appears that one can strongly presume the ability of muramyl-peptides, when administered in combination with a cytokine
- to potentiate the biological activity of the
cytokine considered also allowing to obtain
the desired effects after administrations
lower doses than those considered
currently as therapeutically necessary.

- not to favor and possibly even inhibit
the in vivo induction by this cytokine of
limiting factors its possibilities
therapeutic applications as it has been
observed in the case of association with INF
and, where appropriate and simultaneously
- to induce in situ production, on the one hand,
inhibitors of these limiting factors, by
example IL-1 RA and especially when the cytokine
consists of an interferon, the soluble receptor
of TNF (STNF-R) and, on the other hand, the secretion
in situ by the competent cells of the system
cytokine lymphoids, for example IL-6 and G
CSF in the case of interferons, which allow
then broadening the spectrum of action of the
cytokine of the administered combination.

The invention therefore relates to any combination using a cytokine and any muramyl-peptide not inducing and even allowing if necessary the inhibition in humans of secretion by its competent cells or the inhibition of their activity as biological mediators. such IL-1 and / or IL-8 and / or
TNF whose rattle is highly likely in the side effects related to the administration of certain cytokines.The ability of muramyl-peptide to potentiate the action of the cytokine while protecting the haste against the induction of cytokines or other harmful mediators can, in each case, be verified by comparative in vivo tests, preferably in humans, involving, on the one hand, the selected cytokine alone and, on the other hand, a combination of this cytokine with the muramyl-selected peptide, and detection and measurement on a comparative basis (e.g. in RIA or ELISA assays using appropriate antibodies) rate of induced cytokines studied. The results that can be obtained are, of course, non-limiting examples in the following examples applied to interferon α exposed below.

 Preferably, the cytokines used will always be cytokines of human origin. It should be emphasized that this expression must be understood as covering, in addition to the natural cytokines of human origin (it is impossible to ignore the difficulties involved in their adequate isolation), the corresponding recombinant cytokines produced by genetic engineering techniques. , that is to say cytokines nevertheless characterized by amino acid sequences identical to those of these natural cytokines, which nevertheless does not exclude equivalent amino acid sequences differing from the previous ones, in particular by substitutions, additions or amino acid deletions resulting in neither substantial changes in the characteristic properties of "identical sequence" recombinant cytokines, nor the appearance of deleterious properties (e.g., antibody inductions in human haste).

dans laquelle le groupe R est un hydrogène ou un groupe méthyle ; X est L-alanyle, L-leucyle, Lisoleucyle, L-valyle, L-thréonyle, L-(N-méthyl)alanyle et R7 et R8 sont indépendamment l'un de l'autre, des groupes hydroxyle, amino, O(CH2)xH avec x = 1, 2, 3, 4 ou 5 ou des groupes peptidiques comprenant de 1 à 3 résidus d'acides aminés, de préférence lévogyres.Preferably the muramyl peptides used in the invention are hydrophilic muramyl peptides. I1 are characterized in particular by the following general formula

wherein the R group is a hydrogen or a methyl group; X is L-alanyl, L-leucyl, Lisoleucyl, L-valyl, L-threonyl, L- (N-methyl) alanyl and R7 and R8 are independently of each other hydroxyl, amino, O ( CH2) xH with x = 1, 2, 3, 4 or 5 or peptide groups comprising from 1 to 3 amino acid residues, preferably levogyrous.

Preferred muramyl-peptides are muramethic (R = CH3, X = L-Ala, R7 = OCH3 and R8 =
NH 2, murabutide (R = CH 3, X = L-Ala, R 7 = OnC 4 H 9 and
R8 = NH2) and finally the muradimetide (R = CH3, X = L-Ala,
R7 = R8 = OCH3).

 Other preferred muramyl peptides are the homologues of MDP, murametide, murabutide and muradimetide, in which the L-alanyl groups are replaced by threonyl groups.

 It goes without saying that this class of muramyl peptides is in no way limiting. Lipophilic muramyl-peptides, such as those described in French Patent No. 8407340, may also be used within the scope of the invention. In general, any muramyl peptide that can be used in human therapy can be used for the constitution of the combination.

 Finally, it is self-evident that all other adjuvant-containing muramyl peptides carrying substituents at positions 1, 4 or 6 of the saccharide group can also be used.

 Finally, it will be noted that the term "combination" does not imply that the selected cytokine and muramyl-peptide are necessarily administered in a hurry or in a simultaneous manner. It also extends to any use or presentation involving their administrations at non-zero time intervals, it being understood, however, that these intervals must be sufficiently short to allow the mutual interactions of the two constituents of the association under the conditions indicated above.

 Other features of the invention will become apparent from the nonlimiting examples described below.

In clinical trials in healthy human volunteers, IFN a 2a (interferon
Roferon Assayed at 3 MU / ml marketed by Roche under the trade name ROFERON A, was administered in combination with muramyl peptides, in particular murametide and murabutide. It has been shown
(a) that the secretion of Neopterin which is the
recognized biological marker signing the activity
Immunostimulatory activity of IFN was very strongly
increased following dose administrations
suboptimal IFN, associated with doses of
Murabutide or Muramétide; it is the same
of the IL-1 receptor antagonist (IL-1 RA)
whose IFN alone weakly induces the synthesis which
is increased considerably by the addition of
muramyl peptides thus showing activity
synergistic between IFN and immunomodulators
synthetic; this is also true for the
soluble TNF receptor (STNF-R)
(b) the subjects receiving the association show
a significant increase in the number of
cells of the white line with respect to
group treated by IFN
(c) it appears in the plasma of the subjects treated
by the association, mediators of immunity
Interleukin 6, G-CSF, while not observed
induction of any of the pyrogenic mediators and
inflammatories, particularly
cytokines IL-1, IL-8 and TNF.

 These findings are based more particularly on the results of the tests that follow.

Healthy subjects undergo a series of tests to demonstrate their ability to be included in the trial. They receive subcutaneously the mixtures described in the following Table I of
Murabutide (MUB) or Muramétide (MUM) with Interferon a 2a (IFN a).

Table I

IFN α
<tb><SEP> A <SEP> MUB <SEP> 105U <SEP> 3 <SEP> x <SEP> 105U <SEP> 106U <SEP> 3 <SEP> x <SEP> 106U <SEP> 6x106 <SEP> O
<tb> B <SEP> MUB
<tb> A <SEP> 35 <SEP> g / Kg <SEP> A <SEP> I <SEP> A <SEP> II <SEP> A <SEP> III <SEP> A <SEP> IV <SEP> A <SEP> V <SEP> Witness <SEP> A <SEP> 35
<tb> A <SEP> 100 <SEP> g / Kg <SEP> A <SEP> VI <SEP> A <SEP> VI <SEP> A <SEP> VIII <SEP> A <SEP> IX <SEP> A <SEP> X <SEP> Control <SEP> A <SEP> 100
<tb> B <SEP> 35 <SEP> g / Kg <SEP> B <SEP> I <SEP> B <SEP> II <SEP> B <SEP> III <SEP> B <SEP> IV <SEP> B <SEP> V <SEP> Witness <SEP> B <SEP> 35
<tb> B <SEP> 100 <SEP> g / Kg <SEP> B <SEP> VI <SEP> B <SEP> VII <SEP> B <SEP> VIII <SEP> B <SEP> IX <SEP> B <SEP> X <SEP> Witness <SEP> B <SEP> 100
<tb> O <SEP> Control <SEP> Control <SEP> Control <SEP> Control <SEP> Control <SEP> Placebo
<tb><SEP> IFN <SEP> 105 <SEP> IFN <SEP> 3 <SEP> x <SEP> 105 <SEP> IFN <SEP> 106 <SEP> IFN <SEP> 3 <SEP> x <SEP> 106 <SEP> IFN <SEP> 6 <SEP> x <SEP> 106
<Tb>
Subjects are monitored for three days following administration of IFN and / or muramyl peptides and all the tests and examinations required to perform a phase I trial are performed.

In particular the following examinations are practiced
Hematological analysis with white blood cell counts at the time of injection and at 6h, 12h, 24h, 36h, 60h and 72h after injection.

 Blood samples are taken before the injection and at 6h, 24h and 48 hours after the injection, to collect the serum for the assays of Neopterin, Interleukin 1.6 and 8 G-CSF, IL-1 RA and TNF-a.

 The leukocyte counts are performed in an automatic COULTER COUNTER cell counter.

Blood samples for serum assays are centrifuged at 4 ° C at 3000 rpm for 10 minutes immediately after collection, and the sera are stored at -20 ° C. The leukocyte counts and assays are
Neopterin and cytokine are performed using commercial kits.

RESULTS OF EXAMINATIONS AND TESTS 1) Side effects
The side effects that were listed during the trials (such as migraine, fever, and joint pain) were very mild. In particular, the influenza-like effects observed at high doses of 3M and 6M IFNs are not increased by the combination with the muramyl peptides although there is a strong increase in the biological activity of 1 IFN and appearance of a modified profile of this activity with synthesis of new biological mediators.

2) Leukocyte counts
Neutrophils are the cells that are most affected by leukopenic treatments.

It is the fall in neutrophil levels that is responsible for the decline of non-specific immune defenses in subjects subjected to radiotherapy or cytotoxic and / or myelotoxic chemotherapy. To simplify the reading, only the neutrophil counts were given at the time when their variation is most noticeable.

Table II
Increase in the number of neutrophils in healthy volunteers after administration of muramyl-peptides-IFN &alpha;

<Tb>

IFN &alpha;<SEP> adminstered <SEP><SEP> Muramyl <SEP> peptide <SEP> administered <SEP> No, <SEP> Count <SEP> of <SEP> mentrophils
<tb><SEP> (Number <SEP> units) <SEP> Compound <SEP> Dose <SEP> (g / Kg) <SEP> tested <SEP>% <SEP> line <SEP> of <SEP> base <SEP> n
<tb><SEP> 0 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 268 <SEP>#<SEP> 43
<tb><SEP> 0 <SEP> Murabutide <SEP> 100 <SEP> 4 <SEP> 279 <SEP>#<SEP> 67
<tb><SEP> 1 <SEP> x <SEP> 105 <SEP> - <SEP> 0 <SEP> 6 <SEP> 143 <SEP>#<SEP> 34
<tb><SEP> 1 <SEP> x <SEP> 105 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 265 <SEP>#<SEP> 61
<tb><SEP> 3 <SEP> x <SEP> 105 <SEP> - <SEP> 0 <SEP> 6 <SEP> 169 <SEP>#<SEP> 47
<tb><SEP> 3 <SEP> x <SEP> 105 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 292 <SEP>#<SEP> 112
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 6 <SEP> 147 <SEP>#<SEP> 42
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 207 <SEP>#<SEP> 46
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 232 <SEP>#<SEP> 78
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 6 <SEP> 144 <SEP>#<SEP> 19
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 252 <SEP>#<SEP> 59
<tb><SEP> 3 <SEP> x106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 255 <SEP>#<SEP> 96
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 6 <SEP> 191 <SEP>#<SEP> 75
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 226 <SEP>#<SEP> 74
<tb><SEP> 6 <SEP> x106 <SEP> Murabutide <SEP> 100 <SEP> 6 <SEP> 298 <SEP>#<SEP> 71
<tb> a: Represent the highest values observed
for 24 hours after administration.

b: average + standard deviation.

3) Assays of Neopterin
The assays are performed on the sera taken at time 0, 24h and 48 hours. An RIA (Radio Immuno Assay) kit (Behring Diagnostic, France) is used. The results are expressed in nanomoles / ml, the normal values vary from 4 to 9 nanomoles / ml.

Table III

<tb><SEP> IFN <SEP>&alpha;
<tb><SEP> Muramyl
<tb><SEP> Peptide <SEP> T <SEP> 0 <SEP> 10-5u <SEP> 3 <SEP> x <SEP> 10 <SEP> u <SEP> 106 <SEP> u <SEP> 3 <SEP> x <SEP> 106 <SEP> u <SEP> 6 <SEP> x <SEP> 106 <SEP> u
<tb><SEP> Placebo <SEP> Control <SEP> IFN <SEP> Control <SEP> IFN <SEP> Control <SEP> IFN <SEP> Control <SEP> IFN <SEP> Control <SEP> IFN
<tb><SEP> 0 <SEP> 0 <SEP> 6.00 <SEP>#<SEP> 0.80 <SEP> 6.03 <SEP>#<SEP> 1.58 <SEP> 5.78 <MS>#<SEP> 2.03 <SEP> 3.26 <SEP>#<SEP> 0.85 <SEP> 3.22 <SEP>#<SEP> 0.73 <SEP> 4.87 <SEP>#<SEP> 2.50
<tb><SEP> 24 <SEP> 5.50 <SEP>#<SEP> 0.40 <SEP> 9.02 <SEP>#<SEP><SEP> 2.75 <SEP> 10.21 <SEP >#<SEP> 2.20 <SEP> 9.20 <SEP>#<SEP> 2.13 <SEP> 14.39 <SEP>#<SEP> 2.58 <SEP> 20.95 <SEP>#<SEP> 3.73
<tb><SEP> 48 <SEP> 5,20 <SEP>#<SEP> 0,40 <SEP> 9,43 <SEP>#<SEP> 2,11 <SEP> 11,20 <SEP>#<MS> 1.36 <SEP> 9.72 <SEP>#<SEP> 2.90 <SEP> 14.80 <SEP><SEP>#<SEP> 2.22 <SEP> 18.73 <SEP>#<SEP><SEP> 2.62
<tb><SEP> AH <SEP> A <SEP> III <SEP> A <SEP> IV <SEP> A <SEP> V
<tb><SEP> MUB <SEP> 0 <SEP> NI <SEP> NI <SEP> 5.23 <SEP>#<SEP> 1.06 <SEP><SEP> 5.06 <SEP>#<SEP><SEP> 6.77 <SEP> 4.26 <SEP>#<SEP> 0.34 <SEP> 5.21 <SEP>#<SEP> 0.73
<tb><SEP> 35 <SEP> g / Kg <SEP> 24 <SEP> 9.78 <SEP>#<SEP> 1.18 <SEP> 13.00 <SEP>#<SEP> 3.62 <SEP><SEP> 17.31 <SEP>#<SEP> 2.03 <SEP> 22.61 <SEP>#SEP> 3.94
<tb><SEP> 48 <SEP> 10.67 <SEP>#<SEP><SEP> 3.41 <SEP> 12.41 <SEP>#<SEP><SEP> 2.90 <SEP> 18.01 <MS>#<SEP> 3.56 <SEP> 21.30 <SEP>#<SEP> 6.25
<tb><SEP> Control <SEP> MUB <SEP> A <SEP> VI <SEP> A <SEP> VII <SEP> A <SEP> VIII <SEP> A <SEP> IX <SEP> A <SEP> X
<tb><SEP> MUB <SEP> 0 <SEP> 6.37 <SEP>#<SEP> 0.99 <SEP><SEP> 5.97 <SEP>#<SEP> 1.78 <SEP> 4 , 91 <SEP>#<SEP><SEP> 0.96 <SEP> 3.83 <SEP>#<SEP><SEP> 0.82 <SEP> 4.14 <SEP>#<SEP> 0.49 <SEP> 5.85 <SEP>#<SEP> 0.68
<tb><SEP> 100 <SEP> g / Kg <SEP> 24 <SEP> 7.33 <SEP>#<SEP> 0.75 <SEP><SEP> 11.30 <SEP>#<SEP> 3 , 64 <SEP><SEP> 12.43 <SEP>#<SEP> 2.67 <SEP><SEP> 20.11 <SEP>#<SEP> 6.73 <SEP><SEP> 19.45 <SEP>#<SEP> 4.09 <SEP> 29.14 <SEP>#SEP> 0.97
<tb><SEP> 48 <SEP> 8.22 <SEP>#<SEP> 1.39 <SEP><SEP> 11.76 <SEP>#<SEP> 4.87 <SEP><SEP> 13, <SEP>#<SEP> 2.75 <SEP> 18.13 <SEP>#<SEP> 6.92 <SEP> 19.18 <SEP>#<SEP> 4.00 <SEP> 28.19 <SEP>#<SEP> 0.92
<tb><SEP> B <SEP> III <SEP> B <SEP> IV <SEP> B <SEP> V
<tb><SEP> MUM <SEP> 0 <SEP> NI <SEP> NI <SEP> NI <SEP> 4.42 <SEP>#<SEP> 0.70 <SEP><SEP> 5, 67 <SEP>#<SEP> 0.70 <SEP><SEP> 4.61 <SEP>#<SEP> 1.20
<tb><SEP> 35 <SEP> g / Kg <SEP> 24 <SEP> 12.48 <SEP>#<SEP> 1.89 <SEP><SEP> 16.56 <SEP>#<SEP> 1 , 89 <SEP> 21.17 <SEP>#<SEP> 2.17
<tb><SEP> 48 <SEP> 12.34 <SEP>#<SEP> 2.41 <SEP><SEP> 14.36 <SEP>#<SEP> 3.47 <SEP> 18.33 <SEP >#<SEP> 2,23
<tb><SEP> Witness <SEP> MUM <SEP> B <SEP> VI <SEP> B <SEP> VII <SEP> B <SEP> VIII
<tb><SEP> MUM <SEP> 0 <SEP> 5.43 <SEP>#<SEP> 0.66 <SEP><SEP> NI '<SEP>NI'<SEP> 5.20 <SEP>#<SEP> 1.95 <SEP><SEP> 6.59 <SEP>#<SEP> 1.62 <SEP> 4.06 <SEP>#<SEP> 0.17
<tb><SEP> 100 <SEP> g / Kg <SEP> 24 <SEP> 7,14 <SEP>#<SEP> 1,00 <SEP> 16,33 <SEP>#<SEP> 2,13 <MS> 20.78 <SEP>#<SEP> 2.90 <SEP> 18.41 <SEP>#SEP> 2.60
<tb><SEP> 48 <SEP> 7.39 <SEP>#<SEP> 1.37 <SEP> 14.49 <SEP>#<SEP> 4.72 <SEP> 20.33 <SEP>#<MS> 4.18 <SEP> 16.10 <SEP>#<SEP> 2.74
<Tb>
It can be seen from this table that at time 0 all subjects have normal levels of Neopterin, that the subjects who received only placebo have a constant rate of Neopterin and that Muramétide, or Murabutide at a dose of 100 g / Kg do not influence the rate significantly.

At the lowest doses of interferon, the combination with muramyl peptides allows significant increases in
Neopterin. At the highest dose, Murabutide administered at 100 g / kg also allows to obtain a significant increase, but much lower than that observed with the association.

4) Assays of the IL-1 Receptor Antagonist
(IL-1 RA)
The assays carried out on the sera of the subjects having received 106 units of IFN a are reported in the following table (Table IV) and we see a very strong effect of the muramyl-peptide and interferon combination (here also kit British Biotechnology Products Ltd .).

Table IV
Amount of IL-1 receptor antagonist (IL-1
RA) present in the serum of healthy volunteers, 6 hours after administration of muramyl-peptides and IFN a.

<Tb>

IFN- &alpha; -administered <SEP><SEP> Muramyl <SEP> Peptide <SEP> administered <SEP> Serum SEP>SEP>SEP> IL-1 <SEP> RA
<tb> (Number <SEP> units) <SEP> Compound <SEP> dose <SEP> (g / Kg) <SEP> (Pg / ml) +
<tb><SEP> 0 <SEP> Murabutide <SEP> 100 <SEP> 496 <SEP>#<SEP><SEP> 326 #
<tb><SEP> 3 <SEP> x <SEP> 105 <SEP> - <SEP> 0 <SEP> 258 <SEP>#<SEP> 113
<tb><SEP> 3 <SEP> x <SEP> 105 <SEP> Murabutide <SEP> 100 <SEP> 10294 <SEP>#<SEP><SEP> 16554 *
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 2093 <SEP>#<SEP> 1278
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 16093 <SEP>#<SEP> 1621 *
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP> 16204 <SEP>#SEP> 1879 *
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 7322 <SEP>#<SEP> 3215
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 30563 <SEP>#<SEP> 23410 *
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 15523 <SEP>#<SEP> 6554
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 66870 <SEP>#SEP> 36554 *
<tb> +: detected 6 hours after administration of
compounds, the rates before administration were
all below 200Pg / ml #: average + standard deviation among 6 volunteers
of the same group *: rates substantially different from the rates induced
by the single IFN a (p <0.05-p <0.005 in the test of
Mann Whitney Rank) 5) Assays for IL-6
The assay is performed by an Elisa assay kit (British Biotechnology Products Ltd.) on sera taken at time 0 and after 6 hours. Numbers express pg / ml, normal values are less than 6 pg.

Table V

<tb> IFN- &alpha; -administered <SEP> Muramyl <SEP> peptide <SEP> administered <SEP> IL-6 <SEP> Serum <SEP> Rates
<tb> (Number <SEP> units) <SEP> Compound <SEP> dose (g / Kg) <SEP> (Pg / ml) +
<tb><SEP> 0 <SEP> Murabutide <SEP> 100 <SEQ> 2.80 # 1.60S
<tb><SEP> 0 <SEP> Murametide <SEP> 100 <SEQ> 4.30 # 1.80
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 2.00 # 1.70
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> E <SEP><SEP> 100 <SEP> 15.00 <SEP> + <SEP> 12.00 *
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP> 30.00 # 30.00 *
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 3.60 # 0.40
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> fi <SEP><SEP> 100 <SEP> 30.00 <SEP> + <SEP> 23.00 *
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP> 31.00 <SEP> + <SEP> 19.00 *
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP> 34.00 # 56.00
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP> 76.00 # 58.00
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP> 107.00 # 72.00 *
<tb>: each group included 6 volunteers except the one
for murametic, which included only 4 +: measured 6 hours after administration;
no rate was detected before administration
the detected compound
S: mean + standard deviation *: rates substantially different from induced rates
by the single IFN a (p <0.05-p <0.005 in the test of
Mann Whitney Rank)
It can be seen from Table V that at all the doses studied Murabutide and Interferon acted synergistically.

6) Assays of G-CSF
These assays were performed using the British Biotechnology Products Ltd. kit. (see table
VI)
Table VI
Amount of G-CSF present in the serum of healthy volunteers before and 6 hours after administration of the muramyl-peptide combination and
IFN a.

<Tb>

IFN- &alpha; -administered <SEP><SEP> Muramyl <SEP> Peptide <SEP> Administered <SEP><SEP> Serum <SEP> Rate of <SEP> G-CSF <SEP> After
<tb> (Number <SEP> units) <SEP> Compound <SEP> dose (g / Kg) <SEP> 0 <SEP> time <SEP> 6 <SEP> hours
<tb><SEP> 0 <SEP> Murabutide <SEP> 100 <SEP>><SEP> 10 <SEP> 18 # 9
<tb><SEP> 0 <SEP> Murametide <SEP> 100 <SEP> 61 # 103x <SEP><SEP> 73 # 115
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP>><SEP> 10 <SEP> 13 # 10
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP>><SEP> 10 <SEP> 58 # 51x
<tb><SEP> 1 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP>><SEP> 10 <SEP> 98 # 81x
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP>><SEP> 10 <SEP> 17 # 21
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP>><SEP> 10 <SEP> 77 # 65
<tb><SEP> 3 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP>><SEP> 10 <SEP> 79 # 83
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> - <SEP> 0 <SEP>><SEP> 10 <SEP> 17 # 26
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murabutide <SEP> 100 <SEP>><SEP> 10 <SEP> 140 # 168
<tb><SEP> 6 <SEP> x <SEP> 106 <SEP> Murametide <SEP> 100 <SEP> 12 # 15 <SEP><SEP> 284 # 425x
<tb> *: each group consisted of 6 volunteers, except the one
who received the muramétide is the only one who does
consisted of only 4 volunteers
X: mean + standard deviation *: rates substantially different from induced rates
by the single IFN a (p <0.05-p <0.005 in the test of
Mann Whitney Rank)
In this case also a synergistic activity of muramyl peptides and IFN is observed to induce G-CSF.

7) Soluble TNF Receptor Assays (STNF-R)
These assays are performed by ELISA in the serum of subjects with 6M IFN units. At this dose, there is already a significant increase in the level of STNF-R but the addition of murabutide allows a significant increase in the secretion of this mediator.

Table VII

<tb><SEP> IFN <SEP> - <SEP>&alpha;<SEP> Murabutide <SEP><SEP> Rate <SEP> of <SEP> sTNF-R <SEP> in <SEP><SEP> serum <SEP> Increase <SEP> net
<tb><SEP> units <SEP> (g / Kg) <SEP> (pg / ml) <SEP> (pg / ml)
<tb><SEP> 0 <SEP> hour <SEP> 6 <SEP> hours
<tb> 0 <SEP> 100 <SEP> 195 + 28 <SEP> 216 + 65 <SEP> 31 <SEP> + <SEP> 37
<tb> 6 <SEP> x <SEP> 106 <SEP> 0 <SEP> 229 # 95 <SEP><SEP> 389 # 95 <SEP><SEP> 159 # 108
<tb> 6 <SEP> x <SEP> 106 <SEP> 100 <SEP> 232 # 35 <SEP><SEP> 576 # 139 <SEP><SEP> 345 # 120 *
<tb> *: significantly different from the rates induced by
IFN-a alone (p = 0.005 Mann Whitney Rank Test) 8) Assays for IL-1, IL-8, TNF-a
These assays were carried out under the same conditions and none of these cytokines could be demonstrated in the sera tested. The kits used are all from British Biotechnology Products Ltd.

The results obtained therefore show the following
A) Notably about interferon:
a) At doses for which he is unable to
significantly change the synthesis rate of
Neopterin when administered alone,
the association of IFN with the Murabutide or the
Muramétide allows to obtain important rates
of this marker. At higher doses of IFN,
the association allows a significant rise
its rate. Neopterin being considered as
one of the main markers of the activity of
interferon at the level of human macrophages,
these results therefore show that the effects of
IFN related to the activation of these cells
can be obtained with reduced doses,
well tolerated; a similar remark applies
IL-1 RA with extremely low levels
after administration of the association.
can even observe that there is an activity
synergistic between IFN and murabutide or muramétide
and that we get a dosed effect,
since approximately a doubling of the rate
IL-1 RA is obtained when the IFN dose is
doubled. The same observation can be made for
STNF-R which is induced by IFN alone but
slightly compared with the rates induced by the combination.

b) The combination IFN with muramyl-peptide
allows to obtain more leukocyte counts
higher than those of the witnesses
c) The Association of Muramyl Peptides with IFN aa
additionally caused the selective secretion of others
cytokines or immune mediators. Indeed,
can be noted in subjects treated with
association, that there are differences
between their serum levels of IL-6,
G-CSF and STNF-R and those treated by
IFN alone or Murabutide alone. However, we
does not observe an increase in IL-1 levels,
IL-8 or TNF a. This selective effect,
the association of muramyl peptides with
Interferon on the secretion of cytokines is
quite unexpected and particularly
interesting. It could not be predicted from
known properties of both components
administered alone. This discovery offers
possibilities of very interesting applications.

B) G-CSF is the cytokine responsible for the differentiation of stem cells to the granulocyte lineage and their regeneration after myelotoxic treatment and IL-6 is also very involved in hematopoiesis. Thus, the possibility of associating with IFN activity exogenously the activities of endogenous G-CSF and IL-6 induced by this activity.
IFN has exogenously administered in a hurry, constitutes an important therapeutic advance.
(a) this association is made under conditions
minimal side effects; doses of IFN
needed are low
(b) it operates under much more
physiological dosages and availability that
if we administered a "cocktail" of cytokines
corresponding
c) lack of secretion of TNF, IL-1 and IL-8
is a particularly favorable element,
especially since there is secretion of STNF-R which
combining with any amounts of TNF
would neutralize its action as would IL-1 RA
in the event of an active amount of IL-1.

This dataset leads to new
therapeutic applications in human medicine
and an extension of the fields of application
existing therapies. In particular
1. all situations where the therapeutic interest
IFN can now be highlighted.

In particular, the invention allows the extension
interferons in this form of associations
to treatments for tumor diseases that do not
are hardly considered so far, essentially
because of the too great relative importance
side effects discussed above. We
can think of chronic myeloid leukemia,
different carcinomas, multiple myeloma,
melanoma, to various leukemias and lymphomas.

The invention makes it possible, thanks to the effect of
stimulation of the production of
therapeutically useful cytokines,
reconstruct at least part of the systems
physiological biological complexes normally
involved in maintaining homeostasis,
thanks to the interaction of their constituents with
other soluble regulating factors or
associated with the cells.

2. In particular, situations involving
a deficit of spontaneous granulocyte lines,
as in the case of syndromes
myelodisplastic, or induced by treatments
medicinal products, in particular based on cytokines.

One of the great benefits of using
the association according to the invention lies in
the protection of haste against actions
leukopeniant of the same cytokines used
only, so that cytotoxic compounds
implementing the appropriate cytokine,
particularly for the treatment of cancers,
infectious diseases, or disabilities
of genetic origin can be prolonged
to allow a more secure healing effect. In
other words, the association allows either a
slowdown in depletion, even a
sufficient restoration of the system
granulocyte (or even a repair of the
marrow) whenever it is affected
by cytotoxic treatment (chemotherapy or
cancer radiotherapy, treatment of AIDS with
AZT) and / or immunosuppressant.
can also be used in situations
according to the invention (cytokine and muramyl
peptide) or the cytotoxic agents used
are different from the cytokine of the association,
concurrently with the cytotoxic treatment of
genre in question, or whenever the
treatment with the cytotoxic agent is
interrupted for the purpose of
restoration of phagocytes, platelets
blood and spinal cord stimulation
bone. Finally, the invention opens the way to a
faster system rebuild
hematopoietic disease in people whose
immune system had been before
inhibited or even destroyed, for example to
allow a bone marrow transplant
or more generally of tissues or organs
alien.

Induction of IL-6 also suggests that
Association with an effect on the
megacharyocytopolesis and so on regeneration
platelets. This has been observed
in some of the subjects that were followed
over two weeks.

C) There is also a significant production of IL-1 RA and STNF-R in the absence of secretion of
TNF, IL-1 and IL-8. This leads to a second component of applications. First, the prevention and treatment of septic shock in which IL-1 and
TNF play a vital role. The other indications are all those where it is necessary to oppose inflammation, as in the case of rheumatoid arthritis or certain autoimmune diseases or diseases induced in the case of marrow, tissue or tissue transplants. organs, by a graft reaction against haste.

 D) Naturally, the combination is equally useful for the treatment of viral diseases such as chronic hepatitis C or B or respiratory viral infections, whether it is a treatment in which the cytokine is directly involved in its anti-inflammatory capacity. -viral (for example, interferon) or substitution treatment or between antiviral treatments that must be interrupted intermittently (for example, inhibition of HIV viral multiplication by AZT or other nucleoside derivatives having a similar antiviral action spectrum).

 In general, the invention therefore relates to any combination meeting the above-mentioned criteria, optionally containing, in addition, a pharmaceutically acceptable vehicle. Without it being necessary to limit it, these pharmaceutical compositions advantageously consist of solutions that can be administered parenterally, in particular subcutaneously, intravenously, or by infusion. The muramyl peptides may be administered orally either alone or in a dosage form ensuring their protection (for example, in capsulation in liposome capsules or microspheres allowing the crossing of the gastric barrier .The same is true for the cytokines used, in particular interferons.

 In the case of the combination interferon and muramyl-peptide, the preferred doses of IFN for an injection will be of the order of 5 x 104 to 5 x 106 (0.05M to 5M) International Units and doses of muramyl -peptide, especially muramétide or murabutide, are of the order of 10 g to 250 Ag / kg.

 For the case of GM-CSF and muramylpeptide combinations, preferred doses will be respectively 1 to 25 g / kg of GM-CSF for 10 g to 250 ssg / kg of muramyl-peptides.

 It goes without saying that the doses indicated above have no value other than that of illustrating the invention. It is, of course, the clinician's duty to determine the doses to be associated with the constituents of the association, depending on the condition of the patient and the condition in which he is suffering.

 The invention also relates to the association with IL-2 which has recognized anti-tumor activities, in particular with respect to metastases of renal carcinoma or other cancers, but whose therapeutic index is extremely low. . The preferred doses for an injection would be from 1 to 10 million Kg / day IL-2 units associated with 10 μg to 250 μg / kg muramyl-peptides.

As additional examples, the invention also relates to the combinations with a muramyl peptide of the indicated genus, IL-6, IFNT and / or TGFP factor (abbreviation of "Transforming
Growth Factor "or" Growth Transformation Factor ") They have been implicated in the negative regulation of IgE immunoglobulins, which are responsible for allergic phenomena, and" negative regulation "means a reduction in production. immunoglobulins IgE class relative to the production of other immunoglobulins including IgA and IgG.Administration of the combination of one or more of these cytokines in combination with a muramyl-peptide allows a modulation of the profile of synthesized immunoglobulins by the cells of the immune system, in a sense favoring the production of IgG and IgA with respect to that of IgE, or even to the detriment thereof.This result shows the possibility of using the combination according to the invention for the treatment of allergies in therapy.

The preferred doses of each of the constituents of the combination, especially when it is a question of parenteral administration, would be in particular the following ones, for approximately from 10 g to 250 g / kg of muramyl-peptide.
from 0.05 M to 6 M IU of IFN7,
from 0.5 g to 100 g / kg of TGFB, and / or
from 0.1 g to 25 g / kg of IL-6.

Finally, it is self-evident that the following claims can only extend their effects to compositions, which would involve, in association with the selected cytokine, a molecule of the muramyl-peptide type which can be used in a human clinical setting or a molecule which exhibits the same. essential biological properties. The following products can be mentioned as examples: Wall-L-Thr-D-isoGln-sn glyceryl dipalmitoyl-Nac-Mur-L-Ala-D-isoGln-L-Ala-2 (1 ', 2 'dipalmitoyl) sn-glycero-3'-phosphorylethylamide (MTP-PE); N2 (Nac-Mur-L-Ala-D-isoGln) N6-stearoyl-L-Lysine (Muroctasine) or MDP-Lys (L18) - NAc-glucosaminyl-NAc-Mur-L-Ala-D-isoGln-L -To the
Glyceryl dipalmitate (DTP-GDP) or NAc-glucosaminyl-NAc-Mur-L-Thr-D-iso-Gln-L-Ala-Glyceryl-ipa-Nac-Mur-L-Thr-D-isoGln; ; - Nac-Mur-D-Ala-D-isoGln-1,2-dipalmitoyl-sn-glycerol - Nac-Mur-D-Thr-D-isoGln-1,2-dipalmitoyl-sn-glycerol - CDM analogues in which the 2nd amino acid, glutamine is replaced by a Norleucine, and which are devoid of pyrogenic activity - Derivatives or analogues (obtained by fractionation or better by synthesis) of bacterial products sufficiently free from toxicity, in particular from pyrogenicity, among which endotoxins, especially monomers or peptidoglycans, capable of activating immunologically competent cells and inducing cytokines in man.

Claims (16)

REVENDICATION8  1. Composition combining, for the purpose of their human therapeutic application, a natural or recombinant cytokine, preferably human, selected from IFN 2, TGFP and IL-6, with a muramyl peptide that is clinically acceptable in human medicine.  2. Composition according to claim 1, characterized in that the muramyl-peptide has the following general formula

 wherein the R group is a hydrogen or a methyl group; X is L-alanyl, L-leucyl, Lisoleucyl, L-valyl, L-threonyl, L- (N-methyl) alanyl and R7 and R8 are independently of each other hydroxyl, amino, O ( CH2) xH with x = 1, 2, 3, 4 or 5.  3. Composition according to claim 1, characterized in that the muramyl-peptide is chosen from one of the following muramyl-peptides - Nac-L-Ala-D-wall isoGln-sn glyceryl dipalmitoyl - Nac-L-Thr-D-wall isoGln-sn glyceryl dipalmitoyl-NAc-Mur-L-Ala-D-isoGln-L-Ala-2 (1 ', 2'dipalmitoyl) -sn-glycero-3'-phosphorylethylamide (MTP-PE) -Nz (Nac- Mur-L-Ala-D-isoGln) N6-stearoyl-L-Lysine (Muroctasine) or MDP-Lys (L18) - NAc-glucosaminyl-NAc-Mur-L-Ala-D-isoGln-L-Ala Glyceryl dipalmitate (DTP-GDP) or NAc-glucosaminyl-NAc-Mur-L-Thr-D-iso-Gln-L-Ala-Glyceryl-dipalmitate-Nac-Mur-L-Thr-D-isoGln; Nac-Mur-D-Ala-D-isoGln-1,2-dipalmitoyl-sn-glycerol-Nac-Mur-D-Thr-D-isoGln-1,2-dipalmitoyl-sn-glycerol-CDM analogues in which the 2 amino acid, glutamine is replaced by a Norleucine, and which are devoid of pyrogenic activity  4. Use for the composition for the production of a composition using a therapeutically useful cytokine in humans and for the treatment of allergies, characterized in that the active principles of this composition are constituted by a muramyl-peptide and a cytokine as claimed in any one of claims 1 to 3.  5. Composition according to any one of claims 1 to 3, characterized in that the cytokine is a CSF, in particular a GM-CSF.  6. Use for the production of a composition employing a therapeutically useful cytokine in humans and for the treatment of a condition whose effects are likely to be reduced, if not eliminated by the administration of said composition to the patient , characterized by its association with a muramyl peptide as defined in any one of claims 1 to 3.  7. Use according to claim 6, characterized in that the cytokine is a cytokine such as interferon, in particular an interferon a likely, in combination with the muramyl-peptide, to induce secretion in situ, in the treated patient. , G-CSF and / or IL-6 and / or IL-1 RA.  8. Use according to claim 6 or 7, characterized in that the combination is intended for the treatment of cancers, infectious diseases or genetic deficiencies that can be compensated by the cytokine used.  9. Use according to claim 6 or 7, characterized in that the combination is intended to induce either a slowdown in depletion, or even a sufficient restoration of the granulocytic system (or even a repair of the spinal cord), that these conditions are of a spontaneous character or that they result from a cytotoxic, radiotherapic and / or immunosuppressive treatment.  10. Use according to claim 8 or 9, characterized in that the combination is intended for treatment using a cytotoxic agent distinct from the cytokine of the combination, concurrently with the cytotoxic treatment of the genus in question, or each time that treatment with the cytotoxic agent is discontinued to allow restoration of phagocytes, platelets, and bone marrow stimulation.  11. Use according to claim 6 or 7, characterized in that the combination is intended for a treatment aimed at promoting a faster reconstitution of the hematopoietic system in persons whose immune system had previously been inhibited, or even destroyed, especially for to authorize a transplantation of bone marrow or, more generally, of allogenic or even xenogeneic tissues or organs.  12. Use according to claim 6 or 7, characterized in that the combination is intended for the treatment of viral diseases, whether it is a treatment in which the cytokine, in particular interferon, is directly involved in its treatment. anti-viral or substitution therapy or between treatments conducted with other antiviral substances that must be interrupted intermittently.  Use according to claim 12, applied to the inhibition of HIV viral multiplication by AZT or other nucleoside derivatives having a similar antiviral action spectrum).  14. Use according to claim 6 or 7, characterized in that the cytokine is an interferon or a CSF, in particular a GM-CSF, and in that the combination is intended for the prevention or treatment of septic shock in which the IL-1 plays a key role in the treatment or prevention of inflammatory processes, as in the case of arthritis or certain autoimmune diseases.  15. Use according to claim 6 or 7, characterized in that the cytokine is an IL-2, and in that the combination is intended for the treatment of cancers.  16. Use according to claim 6 or 7, to overcome the effects of inducing drugs or infections inducing an endogenous synthesis of IL-1, IL-8 or TNF.