WO2008135419A2

WO2008135419A2 - Anti-il-6-agents for improving primary functioning of allogenic grafts

Info

Publication number: WO2008135419A2
Application number: PCT/EP2008/055124
Authority: WO
Inventors: Birgit Sawitzki; Weihua Gong; Andreas Pascher; Hans-Dieter Volk
Original assignee: Charite Universitaetsmedizin Berlin
Current assignee: Charite Universitaetsmedizin Berlin
Priority date: 2007-05-04
Filing date: 2008-04-25
Publication date: 2008-11-13
Anticipated expiration: 2009-11-04
Also published as: WO2008135419A3

Abstract

The present invention relates to the use of interleukin-6 antagonists for preventing, treating or ameliorating delayed graft function and/or for improving primary and/or long term graft function, particularly in kidney transplantation, and corresponding medicaments.

Description

Anti-IL-6-Agents for improving primary functioning of allogenic grafts

The present invention relates to the use of interleukin-6 antagonists for preventing, treating or ameliorating delayed graft function and/or for improving primary and/or long-term graft function, particularly in kidney transplantation, and corresponding medicaments.

In organ transplantation, delayed graft functioning is a frequently occurring problem. Particularly in the field of kidney transplantation, it is frequently observed that a transplanted organ is not or not sufficiently functioning within the first days after surgery. This results increased serum creatinine concentration and oliguria, necessitating a dialysis treatment of the organ recipient. Approximately 20 to 50 % of all kidney transplantations are plagued by delayed organ function. It is to be noted that the delayed graft function is not to be confused with graft rejection. Within the scope of the present invention, "delayed graft functioning" is defined as the medical condition after organ transplantation characterized by the absence or incompleteness of the respective organs functions compared to a transplant not suffering from this condition. Particularly, delayed graft function in kidney transplantation is characterized by a lack of decrease in serum creatinine levels compared to the level before surgery. Sometimes, serum creatinine levels even rise post-operatively in delayed graft function.

The reasons for delayed graft functioning are presently not clear. Without being bound by any theory, inventors believe that a weight difference between donor and recipient is a factor augmenting the incidence of delayed graft functioning. Other factors recognized in literature are cold and warm ischaemia time, donor age and overall graft quality.

A number of prophylactic and therapeutic remedies have been suggested particularly in view of kidney transplantation to prevent, treat or ameliorate delayed graft function. Among preventive means are: Application of preventing solutions like the so-called University of Wisconsin solution, application of Vasodilators, particularly calcium channel blockers and atrial natriuretic peptide, antioxidants like heme oxygenase 1 (HO1 ) inductors, and anti-inflammatory medicaments, particularly anti-TNFα antibodies, chemokine antagonists, corticosteroids and toll-receptor, antagonists. Among therapeutic treatments, dialysis and change of immunosuppressive therapies are frequently applied, wherein the standard applications of anti-inflammatory medicaments are, e.g. anti-interleukin-2 receptor antibodies and corticosteroids.

Although a preventing solution containing a steroid, in particular prednisolone, as the active ingredient may improve primary graft function, in addition to common side effects of steroid treatment excessive cell death in the transplanted organ has been observed (see Fig. 6d), which leads to long-term damages of the transplanted organ and, thus, to a decreased long-term survival.

Despite these preventive and therapeutic measures, incidence of delayed graft function is still considerably high (cf. Daly et al., "Delayed graft function: a dilemma in renal transplantation", BJU International (2005), 498 to 501 ; Perico et al., "Delayed graft function in kidney transplantation", Lancet, 3641814-27). It was therefore an object of the present invention to provide improved means for preventing, treating or ameliorating delayed graft function and/or for improving primary and/or long-term graft function with decreased side effects. It was a particular problem of the invention that these means should be applicable in the field of kidney transplantation. The present invention is based on the unexpected discovery that the administration of an interleukin-6 antagonist prevents or counteracts against delayed graft function and also provides a better long-term survival rate without the common side effects correlated with a steroid treatment in comparison with a treatment with prednisolone (see Example 3). The inventive administration is particularly suitable for preventing or treating delayed graft function in kidney transplantation. Upon administration of an interleukin-6 antagonist functioning of a transplanted kidney could be obtained or restored and maintained over a long-term period (measured by serum creatinine concentration; cf. examples).

Therefore, the invention provides the use of an interleukin-6 antagonist in the preparation of a medicament for preventing, treating or ameliorating delayed graft function. The invention also provides the use of an interleukin-6 antagonist in the preparation of a medicament for improving primary and/or long term graft function.

The use of interleukin-6 antagonists had so far only been contemplated in the field of graft rejection (WO 2004/039826). However, delayed graft functioning is different from graft rejection as indicated above; prophylactic and therapeutic approaches for the prevention of graft rejection are not applicable to the field of preventing, treating or ameliorating delayed graft function. Besides, effectiveness of interleukin-6 antagonists for the prevention of graft rejection has so for not been proven but only specu- lated. Particularly WO 2004/039826 does not disclose any indication of the interleukin-6 antagonists described therein for the actual therapeutic effectiveness regarding the treatment or prevention of graft rejection. Instead, this publication only provides a long list of immune conditions, which the authors speculate might require the application of interleukin-6 antagonists. Further interleukin-6 antagonists and their uses are disclosed in WO 96/17869, WO 96/18648, EP 0 936 923 B1 and EP 0 972 780 A1 , again without any indication that interleukin-6 antagonists could be useful at all in preventing, treating or ameliorating delayed graft function, particularly in renal transplantation. Aforementioned publications are incorporated herein by reference in so far as they disclose interleukin-6 antagonists and methods for producing the same.

According to the invention, the interleukin-6 antagonist is typically a small molecule and/or a peptide. In a preferred embodiment the interleukin-6 antagonist is a peptide or more preferably a monoclonal and polyclonal anti-interleukin-6 antibody, chimaeric - A -

anti-interleukin-6 antibody and antigen-binding fragment thereof or monoclonal or polyclonal anti-interleukin-6-receptor antibody and antigen-binding fragment thereof or a peptide derived from the acidic domain of gp130, e.g. 18AD. Suitable anti- interleukin-6 antibodies are disclosed, e.g., in WO 2004/03928621. According to the invention, an anti-interleukin-6-receptor antibody that interferes with the receptor's ability to elicit a normal reaction in response to interleukin-6 exposure is also considered an interleukin-6 antagonist.

In an alternative preferred embodiment the interleuin-6-antagonist is a small molecule selected from the group consisting of galiellalactone; (+)-madindoline; hydrophobic bile acids, preferably glycochenodeoxycholate; Cyclopentenone; 15-deoxy- Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)); and pyrrolidine dithiocarbamate (PDTC). More preferred are galiellalactone and (+)-madindoline. Galiellalactone inhibits the interleukin-6 signal transduction and (+)-madindoline prevents homodimerization of the interleukin-6 receptor.

Advantages of small molecules as interleukin-6 antagonists over peptides as interleukin-6 antagonist may be the reduced risk of immunologic reaction in the recipient and better diffusion properties.

Accordingly, particularly preferred is the use of an interleukin-6 antagonist, wherein the interleukin-6 antagonist is according to the invention a peptide, preferably a monoclonal or polyclonal anti-interleukin-6 antibody, chimaeric anti-interleukin-6 antibody and antigen-binding fragment thereof or monoclonal or polyclonal anti- interleukin-6-receptor antibody and antigen-binding fragment thereof or a peptide derived from the acidic domain of gp130, e.g. 18AD, and/or wherein the interleukin-6 antagonist is a small molecule, preferably a small molecule selected from the group consisting of galiellalactone; (+)-madindoline; hydrophobic bile acids, preferably glycochenodeoxycholate; Cyclopentenone; 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)); and pyrrolidine dithiocarbamate (PDTC) and more preferably galiellalactone and (+)-madindoline in the preparation of a medicament for preventing, treating or ameliorating delayed graft function in renal transplantation.

The invention also provides an interleukin-6 antagonist according to the invention for use in a method for preventing, treating or ameliorating delayed graft function and/or in a method for improving primary and/or long term graft function, wherein the inter- leukin-6 antagonists is a peptide, preferably monoclonal and polyclonal anti- interleukin-6 antibody, chimaeric anti-interleukin-6 antibody and antigen-binding fragments thereof or monoclonal or polyclonal anti-interleukin-6-receptor antibody and antigen-binding fragments thereof or a peptide derived from the acidic domain of gp130, e.g. 18AD, and/or wherein the interleukin-6 antagonist is a small molecule, preferably a small molecule selected from the group consisting of galiellalactone; (+)- madindoline; hydrophobic bile acids, preferably glycochenodeoxycholate; Cyclopen- tenone; 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)); and pyrrolidine di- thiocarbamate (PDTC) and more preferably galiellalactone and (+)-madindoline.

Correspondingly, the invention provides a pharmaceutical composition comprising, consisting essentially or consisting of an interleukin-6 antagonist as described according to the invention and a pharmaceutical acceptable excipient. The interleukin-6 antagonist can also be formulated or used as a mixture of two or more of such indi- vidual interleukin-6 antagonists. Preferably, polyclonal antibodies against interleukin-6 and/or its receptor are considered to be "an interleukin-6 antagonist" according to the present invention.

According to the invention, there is also provided a method for preparing a graft organ and particularly a kidney for transplantation comprising or consisting of the following steps:

providing the organ to be implanted, particularly preferred a kidney, and

administering an interleukin-6 antagonist as described above or a pharmaceutical composition as described above to said organ.

Preferably, the interleukin-6 antagonist, which, as described above, preferably is or comprises an antibody or antigen-binding fragment thereof, is preferably administered outside of the recipient body to the organ, especially the kidney. The method for preparing a graft organ is particularly preferred when comprising or consisting of the following steps:

a) providing an explanted organ to be implanted , preferably a kidney,

b) perfusing the explanted organ, preferably kidney with an effective amount of an interleukin-6 antagonist according to the present invention or a pharmaceutical composition according to the present invention prior to closing blood vessels of the organ, preferably kidney and/or

c) closing the blood vessels of the explanted organ and placing the organ with closed blood vessels in a perfusion solution comprising an effective amount of interleukin-6 antagonist according to the present invention or a pharmaceutical composition according to the present invention.

Preferably the perfusion solution is a cooled solution used during the cold ischemia time.

The effective amount of the interleukin-6 antagonist or a pharmaceutical composition according to the present invention is an amount of the interleukin-6 antagonist, which reduces the activity of interleukin-6 and/or reduces the activity of interleukin-6 receptors. In a preferred embodiment the interleukin-6 antagonist is provided in such an amount that after application all interleukin-6 receptors are blocked.

In a further embodiment of the present invention a method of preventing, treating or ameliorating delayed graft function and/or improving primary and/or long term graft functioning in a mammal is provided, comprising or consisting of the step of administering to a mammal prior, during and/or after implantation of an organ to be implanted, preferably a kidney, an effective amount of an interleukin-6 antagonist of the present invention. In case the interleukin-6 antagonist of the present invention is administered to the mammal (recipient) after the implantation of the organ, then the interleukin-6 antagonist is preferably administered until the primary graft function in the recipient is observed. Thus, the administration period of the interleukin-6 antagonist of the present invention is dependent on the response of the recipient, which can be measured by the serum creatinine levels and/or the glomuleric filtration rate. In a preferred embodiment of the method of preventing, treating or ameliorating delayed graft function, the interleukin-6 antagonist is administered /^'. v. , more preferably /^'. v. via Vena portae.

The invention is described in more detail hereinafter, without limiting the scope of the claims.

The accompanying figures show:

Figure 1 : a correlation between donor and recipient weight difference and serum creatinine levels

Figure 2: a correlation between donor and recipient weight difference and intra- graft interleukin-6 levels;

Figure 3: a correlation between donor and recipient weight difference and intra- graft heme oxygenase 1 levels

Figure 4: serum creatinine levels with low and high weight differences between donor and recipient, and influence of anti-interleukin-6 antibodies on serum creatinine levels.

Figure 5: Serum creatinine levels of recipients 24 hours post-transplantation of allogenic kidney grafts comparing anti-IL6 and steroid treatment

Figures 6a,: Histological analysis (H&E) of grafts harvested 24 hours post- b, c and d transplantation of allogenic kidney grafts.

Examples:

Working Example 1 : Use of interleukin-6 antagonists for graft function tests with donor and recipient weight difference

6 Lewis rats with commensurate-weight of donor rats (-20 to +20% of recipient; "L- WD") and 6 Lewis rats with lower-weight DA donor rats (-25 to -60% of recipient body weight; "H-WD") were used for kidney transplantation experiments. One day after transplantation, primary graft function was determined by serum creatinine analysis, lntragraft interleukin-6 and heme oxygenase 1 gene expression levels were studied using qPCR by standard methods, lntragraft interleukin-6 and heme oxygenase 1 protein expression was analyzed by immunohistochemistry. An additional group of Lewis recipients receiving grafts from low weight donors (-40 to -60% of recipient body weight; "H-WD+anti-IL-6-Rα") were once treated with a 500 μg neutralizing anti- IL6 receptor antibody (clone D7715A7, Biozol) intravenously immediately after operation.

Figure 1 shows that a direct correlation between the weight difference between donor and recipient (L-WD = -20 to + 20 wt%; H-WD = -20 to - 100 wt%) and the serum creatinine levels exists.

Figures 2 and 3 show that in transplantations with a high weight difference between donor and recipient, intragraft interleukin-6 and heme oxygenase 1 levels were in- creased compared to the control group with low weight difference between donor and recipient.

Figure 4 shows that the serum creatinine level in the high weight difference recipients was significantly higher (163.87±62.39) than that in the low weight difference recipients (50.175±8.48, p=0.007), which reflects closely a decreased early graft function in the high weight difference recipients. Interleukin-6 and heme oxygenase 1 gene expression in grafts from lower-weight donor is 10 times higher than that from little weight difference grafts. Immunohistochemistry analysis revealed a dense staining of heme oxygenase 1 and interleukin-6 in tubular epithelial cells of grafts from lower weight donors (no figures). Immediate post-transplatation targeting of IL-6 receptor with 500μg Lv. of an interleukin-6 receptor antagonist ("anti-IL-6-Rα") rescued primary graft function resulting in low serum creatinine levels (82.57±30.63, p=0.019), as indicated by the data shown in column "H-WD+anti-IL-6-Rα". It is thus to be noted that one perioperative application of the 500 μg anti-interleukin-6 receptor alpha chain antibody resulted in a marked reduction of serum creatinine concentration and thus to a re-establishment of primary function.

Using an interleukin-6 antagonist, particularly a monoclonal or polyclonal anti- interleukin-6 antibody or antigen-binding fragment thereof, is thus an effective way to prevent, treat or ameliorate delayed graft functioning resulting from high weight differences between donor and recipient (especially for donor weighing -25 to -60% of the recipient).

Working Example 2: Use of an interleukin-6 antagonist and a stereoid for primary graft function tests with donor and recipient weight difference

2a) Serum creatinine levels:

Figure 5: Recipients from low weight difference (Low WD) combinations showed no signs of delayed graft function as the median serum creatinine level was below 50μmol/l. In contrast recipients from high weight difference (High WD) combinations suffer from delayed primary kidney graft function with significantly increased serum creatinine levels. In recipients from High WD combinations receiving either 500 μg Lv. of an anti-IL6 receptor antibody or 15 mg/kg prednisolone primary graft function was nearly completely restored (Median serum creatinine: anti-IL6R, 76,8μmol/l; prednisolone, 53,1 μmol/l).

The above tests show that both an anti-interleukin-6 receptor antibody as the interleukin-6 antagonist of the present invention and a prednisolone as a steroid rescue in high weight difference organ transplantations primary graft function. 2b) Histological analysis:

In Figure 6b grafts from low weight difference (Low WD) combinations show no signs of tubuli destruction and necrosis of tubular epithelial cells. In contrast thereto, in Figure 6a grafts from high weight difference (High WD) combinations are character- ised by severe tubuli atrophy, destruction and cell necrosis. Figure 6c shows that in recipients from High WD combinations receiving 500 μg i.v. of an anti-interleukin-6 receptor antibody tubular damage was almost completely prevented, whereas grafts of prednisolone treated recipients (15 mg/kg) showed still signs of tubular necrosis (Figure 6d).

Thus, in contrast to the inventive treatment with an interleukin-6 antagonist the steroid treatment cannot prevent tubular epithelial damage leading to defects of the transplanted organ which may cause in long term a new transplantation of the organ within a year.

Working Example 3: DNA microarrav analysis of grafts harvested 24 hours post- transplantation of allogenic kidney grafts

In order to investigate the underlying mechanisms of interleukin-6 antagonist and in particular interleukin-6 receptor antibody antagonist (anti-IL6R antibody) mediated rescue of primary graft function, we have performed gene expression analysis of graft RNA samples using Agilent whole rat genome Oligo Mciroarrays (one-colour).

The following samples were hybridized:

-2 samples from Low WD combinations

-2 samples from High WD combinations

-2 samples of High WD combinations receiving anti-IL6R antibody

-2 samples of High WD combinations receiving prednisolone treatment. The final aim was to identify genes which are differentially affected in their expression by either treatment, i.e. meaning to identify genes which are selectively down- regulated by anti-IL6R or induced by prednisolone treatment.

First results from this test show that in contrast to the inventive treatment with anti-IL- 6R the prednisolone treatment leads to selective induction of genes regulating cell apoptosis and calcium metabolism. Thus, targeting the IL-6 receptor with the inventive interleukin-6 antagonist is more specific in preventing the induction of inflammatory genes and does not influence genes associated with the common side effects observed along with steroid (prednisolone) treatment.

Claims

Patent Claims:

1. Use of an interleukin-6 antagonist in the preparation of a medicament for preventing, treating or ameliorating delayed graft function and/or for improving primary and/or long term graft function.

2. Use according to claim 1 , wherein the interleukin-6 antagonist is a peptide or a small molecule.

3. Use according to claim 1 or 2, wherein the interleukin-6 antagonist is a mono- clonal or polyclonal anti-interleukin-6 antibody and antigen-binding fragment thereof or a monoclonal or polyclonal anti-interleukin-6-receptor antibody and antigen-binding fragment thereof or a peptide derived from the acidic domain of gp130, e.g. 18AD.

4. Use according to claim 2, wherein the small molecule is selected from the group consisting of galiellalactone; (+)-madindoline; hydrophobic bile acids, preferably glycochenodeoxycholate; Cyclopentenone; 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)); and pyrrolidine dithiocarbamate (PDTC). More preferred are galiellalactone and (+)-madindoline.

5. Use according to anyone of claims 1 to 4, wherein the medicament is for preventing, treating or ameliorating delayed graft function and/or for improving primary and/or long term graft function in renal transplantation.

6. Interleukin-6 antagonist for use in a method for preventing, treating or ameliorat- ing delayed graft function and/or for improving primary and/or long-term graft function.

7. Interleukin-6 antagonist according to claim 6, wherein the interleukin-6 antagonist is a peptide or a small molecule.

8. Interleukin-6 antagonist according to claim 7, wherein the interleukin-6 antagonist is a monoclonal or polyclonal anti-interleukin-6 antibody and antigen-binding fragment thereof or a monoclonal or polyclonal anti-interleukin-6-receptor antibody and antigen-binding fragment thereof or a peptide derived from the acidic domain of gp130, e.g. 18AD.

9. lnterleukin-6 antagonist according to claim 7, wherein the small molecule is selected from the group consisting of galiellalactone; (+)-madindoline; hydrophobic bile acids, preferably glycochenodeoxycholate; Cyclopentenone; 15-deoxy- Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)); and pyrrolidine dithiocarbamate (PDTC). More preferred are galiellalactone and (+)-madindoline.

10. Pharmaceutical composition, comprising, consisting essentially or consisting of an lnterleukin-6 antagonist and a pharmaceutically acceptable excipient.

1 1. Method for preparing a graft organ for implantation, comprising the steps of

providing the organ to be implanted, and administering an lnterleukin-6 antagonist of any of claims 6 to 9 or a pharmaceutical composition of claim 10 to said organ.

12. Method according to claim 1 1 , comprising or consisting of the following steps:

a) providing an explanted organ to be implanted , preferably a kidney,

b) perfusing the explanted organ, with an effective amount of an interleukin-6 an- tagonist according to any of claims 6 to 9 or a pharmaceutical composition of claim 10 prior to closing blood vessels of the organ, preferably kidney and/or

c) closing the blood vessels of the explanted organ and placing the organ with closed blood vessels in a perfusion solution comprising an effective amount of an interleukin-6 antagonist according to any of claims 6 to 9 or a pharmaceuti- cal composition of claim 10.

13. Method of preventing, treating or ameliorating delayed graft function and/or improving primary and/or long-term graft function comprising the step of administering to a mammal prior, during and/or after implantation of an organ to be implanted, preferably a kidney, an effective amount of an interleukin-6 antagonist according to any of claims 6 to 9 or a pharmaceutical composition of claim 10.