Labelled elastase inhibitors

Details Labelled elastase inhibitors Labelled elastase inhibitors

2000-05-16

2002-04-23

Jones, Dameron L. (Department: 1619)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

In an organic compound

C424S001110, C548S952000

Reexamination Certificate

active

06375926

ABSTRACT:

This a 371 of PCT/GB97/02467, filed on Sep. 10, 1997.
The present invention relates to a class of compounds useful in the diagnosis or radiotherapy of infection, inflammation or thrombi, pharmaceutical formulations containing them, their use in diagnosis of disease and methods for their preparation.
Diagnostic imaging of infection or inflammation in clinical practice typically uses either the radiopharmaceutical
67
Ga citrate, or radiolabelled white blood cells (leucocytes) since leucocytes are known to accumulate at sites of infection/inflammation.
111
In or
99m
Tc are the radioisotopes normally used to label leucocytes.
Labelled leucocytes have become the method of choice in current clinical practice for the diagnostic imaging of sites of infection/inflammation. With the favourable imaging characteristics of
99m
Tc, the specificity of labelled leucocytes and good background clearance, this approach lends itself to the diagnosis of gut lesions such as inflammatory bowel disease or appendicitis. Such diseases cannot be diagnosed with non-specific agents due to the high gut background levels. The problem with the ex-vivo labelled leucocyte approach is that the current cell labelling agents used are non-specific. This means that the leucocytes have to be first separated from the excess red blood cells in blood taken from the patient to be imaged. The cell separation is a labour-intensive operation which requires a skilled operator to achieve efficient separation without compromising white cell integrity. There is also the intrinsic hazard associated with manipulation of blood samples. There has therefore been considerable interest in the field in developing an agent which could be used to image sites of infection which does not require this onerous cell separation prior to labelling and administration to the patient.
Human leucocyte elastase (HLE) is a powerful endopeptidase enzyme capable of hydrolysing amide bonds in a variety of proteins and peptides, including the structural proteins elastin, collagen and fibronectin [R L Stein et al, Ann. Rep. Med. Chem., 20, 237 (1985); P D Edwards and P R Bernstein, Med. Chem. Rev., 14, 127 (1994)]. At sites of infection or inflammation HLE is released by the activated leucocytes and causes tissue destruction. It is also known that leucocytes accumulate in thrombi and that elastase is released during blood coagulation. The released elastase has been shown to be important in fibrinogenlysis [E. F. Plow, J. Clin. Invest., 69, 564-572 (1982)].
EP 0595557 A1 (Merck) discloses that the following compounds are useful as HLE inhibitors for the treatment of inflammatory pathologies such as emphysema or rheumatoid arthritis:
where: R is C
1-6
alkyl;
R
1
is C
1-6
alkyl or C
1-6
alkoxy-C
1-6
alkyl;
M is H, C
1-6
alkyl, hydroxyalkyl, alkoxyalkyl, haloalkyl or C
2-6
alkenyl;
Ra and Rb are H, Hal, OH, Ph, COOH or C
1-6
alkyl, alkoxy or ester;
R
2
and R
3
are H, Hal, COOH, Ph, OH, CN, amino, C
1-6
alkyl, alkoxy or ester, or amide
R
4
is amide or ester with an optional alkyl spacer group.
Radio labelled HLE inhibitors have been little studied. Moser et al. [Am. J. Med., 84 (Suppl.6A), 70 (1988)] report that
131
I-labelled &agr;
1
-antitrypsin persists in the human lung for up to one week post injection with little or no uptake in the liver or spleen. The paper does not, however, discuss potential applications of radiolabelled (&agr;-antitrypsin. A tritium-labelled synthetic HLE inhibitor has been used to study the pharmacokinetics of the HLE inhibitor in rats and monkeys [J. B. Doherty et al, Proc. Nat. Acad. Sci. USA, 90, 8727-31 (1993)]. A
13
C-labelled synthetic HLE inhibitor has been prepared to study the interaction of the inhibitor with HLE in vitro [B. G. Green et al, Biochem. 34, 14331-14355 (1995)]. Neither publication discloses the use of labelled HLE inhibitors for diagnostic imaging of infection, inflammation or thrombi nor are
3
H or
13
C suitable labelling moieties for external diagnostic imaging. Rusckowski et al [J. Nucl. Med., abstract P667 (1996)] report that a genetically engineered protein inhibitor of HLE named EPI-hNE-2 (molecular weight 6759) can be radiolabelled with
99m
Tc via a bifunctional chelate. The radiolabelled protein is reported to show some uptake in a mouse infection model. EPI-hNE-2 is an oligopeptide and could suffer from the same immunogenic problems as Fab′ or larger fragments of antibodies. Furthermore, the size of the molecule limits migration across cell membranes (e.g. those of granulocytes), hence intracellular HLE would not be targeted using this approach.
Blaszczak et al [J. Lab. Comp. Radiopharm., 27, 401-406 (1989)] have described the preparation of
125
I-radiolabelled penicillin V (i.e. a bicyclic &bgr;-lactam) for use in the in vitro assay of penicillin binding protein. There is no suggestion in the paper of in vivo imaging applications and
125
I would not be a preferred radioisotope for external imaging.
It has now been discovered that labelled synthetic HLE inhibitors are useful in the detection of sites of infection or inflammation. Use of a synthetic as opposed to a proteinaceous or polypeptide inhibitor has the significant advantages that the chemical nature of the agent can be fully defined, and potential concerns over immunogenicity are avoided. In addition the position of the label is known unambiguously, and unlike chemotactic peptides or interleukins, the labelled elastase inhibitor is not required to be of very high specific activity because there is an excess of elastase present both within granulocytes and at sites of infection/inflammation. The labelled HLE inhibitors are also useful in the detection of thrombi.
Thus the present invention relates to diagnostic agents for the detection of sites of infection or inflammation or thrombi in the human body. The agents comprise a synthetic human leucocyte elastase (HLE) inhibitor which has a molecular weight of less than 2000 Daltons and is labelled with a detectable moiety suitable for external imaging (e.g. by scintigraphy or MRI), such as a radionuclide or a paramagnetic metal ion. The agent acts by targeting HLE either within leucocytes (in vivo or in vitro), or at sites of HLE release such as sites of infection, inflammation or thrombi. Radiolabelled HLE inhibitors have been shown to selectively label human granulocytes in vitro and to target sites of infection/inflammation in vivo in an animal model of this pathology.
The “detectable moiety” is a substance suitable for external imaging after human administration such as a radionuclide which emits radiation that can penetrate soft tissue; a paramagnetic moiety as a contrast agent for MRI (e.g. certain metal ions such as gadolinium(III), or manganese(II)); a radiopaque moiety such as lopamidol for X-ray contrast imaging (computer assisted tomography ) or an ultrasound contrast agent. Preferably, the detectable moiety is a radionuclide which is either a positron emitter (such as
18
F,
11
C,
15
O,
13
N,
68
Ga or
64
Cu) or a &ggr;-emitter such as
123
I,
99m
Tc,
111
In,
113
m In or
67
Ga. Most preferred radionuclides are &ggr;-emitters, especially
123
I and
99m
Tc.
3
H and
14
C do not have radioactive emissions suitable for external imaging and are therefore outside the scope of the present invention. It is also envisaged that certain radionuclides will confer useful radiotherapeutic properties on the labelled HLE inhibitors. Thus for example
90
Y,
89
Sr,
186
Re,
188
Re,
125
I or
131
I labelled HLE inhibitors could be used in the treatment of rheumatoid arthritis and other bone infections/inflammations. In such applications the therapeutic effect would be due to the local targeted radioactive dose delivered to specific cells, as opposed to any pharmacological effect due to the inhibitor. Whichever detectable moiety is chosen, it is strongly preferred that it is bound to the synthetic HLE inhibitor in such a way that it does not undergo facile metabolism in blood (in vivo or in

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