Description

The invention relates to a further medical use of the polyglycerol derivative, namely to its use for diagnosing a disease at an early stage of the disease by site-specific imaging of angiogenesis, of extracellular matrix remodeling and/or of activated lymph nodes in a human or an animal, wherein the early stage of the disease is characterized by the absence of obvious clinical symptoms due to the disease according to the preamble of claim 1 .

Early diagnosis of diseases is an important field of application in the medical product industry. Today, medical professionals may use in-vitro diagnostics and imaging diagnostics, as well. While in-vitro diagnostics provide a fast and reliable information on the nature and the extent of a disease, they are usually not well suited for diagnosis of a disease at a very early stage.

With respect to early disease diagnosis, medical imaging procedures are well established in clinical routine and will be carried out more than 800 million times per year. The majority of examinations are ultrasound examinations, X-ray procedures such as computer tomography (CT), and magnetic resonance imaging (MRI). During the last two decades, an increasing number of optical procedures and methods applying radioactive isotopes are performed. Furthermore, early diagnosis is crucial in the diagnosis of chronic disease in order to exacerbate disease progression and maintain organ functions. However, diagnostic imaging alone often only provides limited information at the early disease stage. For example, screening mammography may allow for early detection of cancer lesions, especially if provided in systematic screening programs. However, a substantial number of cancer cases are so far only diagnosed at a late stage of disease. The main reason for the under-diagnosed early disease stage is the low specificity.

One approach to improve disease diagnosis at a very early stage of disease is the application of diagnostic imaging agents aimed to improve the diagnostic signal or to modulate the signal. Today, routine diagnostic imaging agents are available for most imaging methods. These imaging agents are selected in such a way that, on the one hand, their application is acceptable in man and that, on the other hand, they can interact with the physical signal of the applied imaging modality in a very specific manner. Diagnostic imaging agents can be divided in two groups: (a) agents containing passive effector molecules, and (b) agents containing active effector molecules. Contrast agents for X-ray belong to the most frequently used group and contain passive effector molecules. These substances comprise a great number of elements with high electron density, such as iodine, which attenuate the applied X-ray energy. A major disadvantage of imaging agents containing passive effector molecules is the high amount of substances needed to achieve a diagnostic contrast information. Application of high dosage is prone to give unwanted pharmacodynamics effects such as impairment of renal function. Moreover, the high amount of passive effector molecules needed to achieve contrast information hamper utilization for early disease diagnosis at a very small lesion size.

Unlike passive effector molecules, active effector molecules such as diagnostic fluorescence dyes for optical imaging or diagnostic radioactive isotopes in nuclear medicine are well suited for early disease diagnosis. They actively emit physical energy, which can be registered using detection systems outside the human body. Due to the high sensitivity of detection systems for isotopes and fluorescence substances in the near infrared spectrum of light, optical imaging methods and nuclear medicine modalities are principally qualified for detection of small lesions. However, a relevant disadvantage of active diagnostic probes is their need for targeting the lesion site. While a number of active effector molecules can be used for imaging of the blood circulation in larger and smaller vessels, the vast majority of substances for optical imaging and nuclear medicine do not accumulate in the lesion and are thus useless to diagnose a disease.

One possibility to enable applicability of active diagnostic effect molecules for early diagnosis of diseases is the chemical conjugation to targeting molecules. These molecules shuttle the active diagnostic effector to the lesion site and leads to accumulation while the unbound effector is cleared from circulation. A relevant limitation in utilizing targeting molecules is the necessity to achieve strong binding to the molecular disease-specific target. In this regard, only macromolecular targeting molecules can be selected to achieve relevant accumulation. Molecules proven for targeting and coupled to active diagnostic probes can be selected from the group of peptides, proteins (e.g. antibodies) and oligonucleotides.

A major disadvantage of macromolecular targeting substances, especially of molecules having a molecular size of > 70 kDa, is the slow clearance from the blood circulation. In most cases, the unbound circulating part of the imaging agent hampers detection of the specifically bound proportion. Another major concern regarding proven macromolecules for targeting of diagnostic effectors is the high cost of goods that oppose economically meaningful product development. There is still an unsolved problem to provide a targeting molecule for reasonable cost of goods, essential targeting properties and fast elimination of the unbound portion from blood circulation. Macromolecular polymers represent another opportunity to shuttle diagnostic probes to target lesions. WO 201 1/09531 1 A1 describes sulfated dendritic polyglycerol conjugated with diagnostic effect molecules for diagnosis of disease. Further information about dendritic polyglycerol sulfate chemically conjugated to the fluorescence dye indocyanine green (ICG) for in-vivo imaging and to indocarbocyanine (ICC) for in-vitro imaging can be obtained from Licha et al. (Licha et al., Bioconjugate Chem. 201 1 , 22, 2453-60). Here, dendritic polyglycerol sulfate-ICG conjugates were utilized for imaging diagnosis of swollen joints in a rat model of rheumatoid arthritis. Licha et al. reports on a failure of dendritic polyglycerol sulfate to diagnose rheumatoid arthritis in joints with disease score 0. This implies that apparently only clinically evident disease can be diagnosed with dendritic polyglycerol sulfate-ICG conjugates.

This conclusion is in line with studies addressing the molecular mechanism of dendritic polyglycerol sulfate in the prior art. In detail, the targeting property of dendritic polyglycerol sulfate is determined by its binding to L- and P-selectin, which are mainly present on circulating blood cells (L-selectin) and activated platelets and vascular endothelia (P-selectin) (Dernedde et al., PNAS 2010, 107, 19679-84).

Further information in the prior art on the diagnostic use of dendritic polyglycerol sulfate-ICG conjugates is provided by studies in ovalbumin-induced asthma in mice (Biffis et al., PLoS One 2013, 8, e57150). Again, established clinical disease can be diagnosed by dendritic polyglycerol sulfate-ICG conjugates that is mainly due to an accumulation of the imaging agent in macrophages.

WO 2008/015015 A2 describes dendritic polyglycerol sulfates and sulfonates and their use for inflammatory diseases. This international patent application also relates to the use of these compounds for imaging diagnostics, in particular with respect to inflammatory diseases however, no indication is provided that the polyglycerol sulfates and sulfonates could be suited for early disease diagnosis.

WO 2009/121564 A1 describes drug conjugates with polyglycerols, wherein a diagnostically active compound can be conjugated to a polyglycerol core. Once again, no indication is provided that such conjugates could be suited in diagnosing early stages of a disease. US 2015/0265731 A1 describes a method of improving tumor diagnostic efficiency of multivalent ligands by regulating the stoichiometric ratio between inner surface functionalities and ligand moieties for tumor targeting, and the multivalent ligands for tumor diagnosis. Summarizing, dendritic polyglycerol sulfate-ICG and dendritic polyglycerol sulfate-ICC conjugates for optical imaging of advanced stages of rheumatoid arthritis, tumors and asthma are known from prior art. There is no hint in prior art that dendritic polyglycerol (sulfate) can be used for early disease diagnosis at a clinical stage without obvious symptoms. It is an object of the present invention to provide a suited possibility to diagnose diseases at an early stage.

Surprisingly, this object could be achieved by providing a further medical use of polyglycerol derivatives as outlined in claim 1. Specifically, the instant invention discloses the further medical use of a polyglycerol derivative for diagnosing a disease at an early stage of the disease by site-specific imaging of angiogenesis, of extracellular matrix remodeling and/or of activated lymph nodes in a human or an animal, wherein the early stage of the disease is characterized by the absence of obvious clinical symptoms due to the disease.. Thereby, the polyglycerol derivative comprises on the one hand a dendritic polyglycerol backbone comprising a plurality of hydroxyl groups that are optionally at least partly substituted by sulfate groups and on the other hand a diagnostic effector residue.

If a classification system for the specific disease to be diagnosed exists, the early stage of the disease typically corresponds to stage 0. It is typically extremely difficult to diagnose a disease with sufficient sensitivity and specificity at an early stage. Since the polyglycerol derivatives according to the instant invention specifically accumulate at sites of angiogenesis and of extracellular matrix remodeling, the diagnostic effector residue conjugated to the polyglycerol backbone (core) of the polyglycerol derivative can be specifically transported to the site of interest. Afterwards, the diagnostic effector residue can be detected resulting in a diagnosis of the disease at an early stage.

The inventors of the present invention have surprisingly found a methodology of diagnosing a disease by targeted imaging of angiogenesis, extracellular matrix remodeling and/or activated lymph nodes by dendritic polyglycerol and/or dendritic polyglycerol sulfate conjugated to diagnostic effector residues (diagnostic effector molecules). The solution of the objective problem according to an aspect of the invention is based on a surprising finding that such dendritic polyglycerol (sulfate) conjugates specifically bind with high affinity to the enzyme heparanase, which is crucial in extracellular matrix remodeling. The binding of dendritic polyglycerol (sulfate) to heparanase enables accumulation of the conjugated diagnostic effector residue at the very first step of disease manifestation in the extracellular matrix of an organ.

Extracellular matrix (ECM) remodeling is a hallmark of many diseases and represents an attractive target for diagnostic agents. The term "remodeling" describes the reorganization of existing extracellular matrix. This process can either change the characteristics of an extracellular matrix such as in blood vessel remodeling, or result in the dynamic turnover of an extracellular matrix such as in bone remodeling. The extracellular matrix is a highly dynamic structure that is present in connective tissue and continuously undergoes controlled degradation and reconstruction. This process involves quantitative and qualitative changes of ECM components and is accompanied by activation of specific proteolytic and carbohydrate degrading enzymes. ECM components interact with cells to maintain homeostasis and therefore regulate diverse functions, including proliferation, migration and differentiation.

The enzyme heparanase is of utmost importance in the early phase of ECM remodeling as it enables sprouting of new capillaries. Heparanase is a 58 kDa heterodimer consisting of a 8 kDa and a 50 kDa protein fragment which cleaves polymeric heparan sulfate. ECM turnover by heparanase permits an invasive program by specialized endothelial cells whose phenotype can be regulated by inflammatory stimuli. Further matrix remodeling and vascular regression contribute to the resolution of the inflammatory response and facilitate extracellular matrix repair. Heparanase also plays an important role in tumor metastasis and regulation of the activity of inflammation. It has been shown that heparanase is a critical component in recruitment of leukocytes to the site of inflammation and mediates leukocyte and mononuclear cell adhesion to endothelial cells.

The inventors could show that targeting heparanase with suited diagnostic effector residues makes it possible to diagnose various diseases at a very early stage before clinical symptoms may become evident. As heparanase activity is indispensable in the process of ECM remodeling, a diagnostic compound against heparanase provides a high sensitivity and specificity regarding ECM remodeling. Additionally, heparanase is also a key enzyme in angiogenesis. Therefore, targeting heparanase with the dendritic polyglycerol (sulfate) conjugates according to the instant invention is also very suited to image those sites with in a body which angiogenesis takes place. It should be noted that angiogenesis is supported by macrophages so that the polyglycerol derivatives according to the instant invention are also well suited for imaging macrophages. Surprisingly and unexpectedly, by the present invention, dendritic polyglycerol (sulfate) conjugates are provided that enable delivery of diagnostically active molecules to the remodeling of the ECM and/or the sites of angiogenesis that are otherwise not capable of targeting ECM and/or the sites of angiogenesis. Moreover, specific targeting of ECM remodeling processes and heparanase is enabled by the dendritic polyglycerol (sulfate) conjugates of the invention. In this context, the dendritic polyglycerol (sulfate) conjugates enable the improvement of the specificity and sensitivity of a broad spectrum of diagnostic compounds, including diagnostic compounds for optical imaging, optoacoustic imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI).

In an aspect, the present invention provides dendritic polyglycerol (sulfate) conjugates for nuclear medicine modalities such as PET and SPECT.

In an aspect, the present invention relates to the further medical use of dendritic polyglycerol (sulfate) conjugates that enable delivery of diagnostically active molecules to activated lymph nodes at early stage of disease. This finding could not be expected from diagnostic imaging utilizing diagnostic effector residues for optical imaging, which only demonstrated diagnostic targeting of the primary disease lesion. Unexpectedly, utilizing dendritic polyglycerol (sulfate) conjugated with isotopes for nuclear medicine, the inventors discovered a targeting of activated lymph nodes distant from the primary lesion site. This finding is an important improvement of diagnostic solutions according to prior art as the exact stage of a disease can now be diagnosed. With respect to treatment, physicians can now detect distant lymph nodes involved in the disease process and consider this finding for treatment planning.

In an embodiment, the animal is a non-human mammal or a rodent.

In an embodiment, this site-specific imaging is additionally or alternatively used to monitor the progression of the disease. Since a disease can already be diagnosed at a very early stage, very fine progress monitoring is also possible by the claimed use of the polyglycerol derivative.

In an embodiment, the exact stage of the disease is alternatively or additionally diagnosed on the basis of the site-specific imaging. Such an exact determination of the stage of the disease helps in providing the best possible therapy to the examined human or animal. While the instantly claimed novel use of a polyglycerol derivative is suited to diagnose many different diseases, this particularly well suited to diagnose myelitis, encephalitis, pneumonitis and/or diseases related to reperfusion injuries. The site-specific imaging is not only suited to finally diagnose diseases, but also to track cells in vivo. Therefore, the instantly claimed use relates in an embodiment to be applied for such tracking of cells in vivo. Such a tracking might also help in diagnosing diseases, depending on the specific disease to be diagnosed and on the cell distribution usually observed upon onset of the according disease.

In an embodiment, the polyglycerol core of the polyglycerol derivative comprises only unsubstituted hydroxyl groups. Thus, in this embodiment, the polyglycerol core is a "pure" polyglycerol. In another embodiment, at least some of the hydroxyl groups of the polyglycerol core are substituted by sulfate groups so that the core of the polyglycerol derivative is a polyglycerol sulfate. Thereby, the degree of sulfation lies in the range between 10 and 100% (including the upper and lower limit). In an embodiment, the degree of sulfation of the backbone is between 15 % and 95 %, in particular between 20 % and 90 %, in particular between 25 % and 85 %, in particular between 30 % and 80 %, in particular between 35 % and 75 %, in particular between 40 % and 70 %, in particular between 45 % and 65 %, in particular between 50 % and 60 %, in particular between 55 % and 58 %, (in each case including the upper and lower limits). Another very well suited degree of substitution is between 20 % and 95 %. Another very well suited degree of substitution is between 30 % and 95 %. Another very well suited degree of substitution is between 50 % and 95 %. Another very well suited degree of substitution is between 65 % and 90 %. Another very well suited degree of substitution is between 90 % and 100 % (in each case including the upper and lower limit). The degree of sulfation can be adjusted by adjusting the experimental conditions under which the sulfation takes place. The diagnostic effector residue is covalently bound to the polyglycerol core to form the polyglycerol derivative. In an embodiment, the diagnostic effector residue is covalently bound to the polyglycerol backbone via a linker or a spacer. Further chemically groups that simplify the attachment of the diagnostic effector residue to the polyglycerol backbone can also be present in the polyglycerol derivative. The average molecular weight of the polyglycerol core according to the present invention is, in an embodiment, 100 to 1 ,000,000 g/mol, in particular 500 to 100,000 g/mol, in particular 2,000 to 50,000 g/mol. Depending on the choice of the polymerization conditions the polyglycerol core reaches a branching degree and an arbitrarily adjustable molecular weight with narrow polydispersities. According to the present invention, polyglycerol cores with a branching degree of more than 0 up to 100 % may be used. In an embodiment, highly branched structures are used, in particular with a branching degree of 20 to 90 %, in particular of 30 to 80 %, in particular of 40 to 70%, in particular of 50 to 60%, in particular having a branching degree of around 60 % (55 % to 65 %).

Depending on the choice of the polyglycerol cores and the sulfation conditions, i.e. the resulting degree of sulfation, the molecular weight of a dendritic polyglycerol sulfate that can be used according to the present invention is, in an embodiment, 200 to 5,000,000 g/mol, in particular 2,000 to 50,000 g/mol, in particular 5,000 to 13,500 g/mol.

A particularly suited embodiment of a dendritic polyglycerol sulfate has a polyglycerol core with an average molecular weight of 2,500 g/mol, a degree of sulfation of 85 % and a molecular weight of 5,500 g/mol.

A further particularly suited embodiment of a dendritic polyglycerol sulfate has a polyglycerol core with an average molecular weight of 5,000 g/mol, a degree of sulfation of 79 % and a molecular weight of 10,500 g/mol.

A further particularly suited embodiment of a dendritic polyglycerol sulfate has a polyglycerol core with an average molecular weight of 2,500 g/mol, a degree of sulfation of 92 % and a molecular weight of 6,800 g/mol. A further particularly suited embodiment of a dendritic polyglycerol sulfate has a polyglycerol core with an average molecular weight of 4,000 g/mol, a degree of sulfation of 84 % and a molecular weight of 8,600 g/mol.

A further particularly suited embodiment of a dendritic polyglycerol sulfate has a polyglycerol core with an average molecular weight of 6,000 g/mol, a degree of sulfation of 76 % and a molecular weight of 12,300 g/mol. In an embodiment, the polyglycerol derivative corresponds to the formula Pn(OS0 ₃ -)x(M ⁺ )x(L-G-D) _m , wherein

P is a dendritic polyol macromolecule,

n is a number of hydroxyl groups of the polyol macromolecule and is > 10,

OSOy is a sulfate group substituting a hydroxyl group of the polyol macromolecule,

M ⁺ is a cationic inorganic or organic counter ion to the anionic sulfate group, x is a number of the sulfate groups and their corresponding counter ion,

wherein 0 < x < n,

D is a diagnostic effector residue,

L is absent or a linker or spacer between P and D,

G is absent or a reactive group for a covalent attachment between L and D, and m is a number of from 1 to 100. In an embodiment, the diagnostic effector residue accounts to less than 50 % by weight to the polyglycerol derivative. In an embodiment, the polyglycerol derivative has a solubility in water of more than 100 mg/ml.

In an embodiment, the linker L is a branched or linear Ci- ₂ o-alkyl group in which one or more non-consecutive methylene groups may be replaced by a group selected from O, S, NH, C(0)NH, C(O), S0 ₂ , SO, aryl, ethene or ethyne, and wherein G is selected from the group comprising -OH, -OS0 ₃ H, -OS0 ₃ ,-NH ₂ , -N ₃ , -COOH, -SH, -S0 ₃ , -C≡C.

Regarding further suited chemical structures of the polyglycerol derivative the use of which is instantly claimed, reference is made to WO 201 1 /09531 1 A1 , the content of which regarding suited polyglycerol derivative structures is herewith incorporated by reference.

In an embodiment, the diagnostic effector residue comprises a dye entity that can be excited by electromagnetic radiation, such as visible light, ultraviolet (UV) radiation or infrared (IR) radiation, in particular near infrared (NIR) radiation. Suited dye entities are, e.g., fluorescence- emitting dyes that are commonly known to a person skilled in the art.

In an embodiment, the diagnostic effector residue comprises a dye entitiy and/or a nanoparticle that can attenuate electromagnetic radiation to enable optoacoustic imaging, such as visible light, ultraviolet (UV) radiation or infrared (IR) radiation, in particular near infrared (NIR) radiation. Suited dye entities are, e.g., fluorescence-emitting dyes that are commonly known to a person skilled in the art. In an embodiment, a suited dye is selected from the group comprising [2-(4-nitro-2,1 ,3- benzoxadiazol-7-yl)aminoethyl] (NBD) dyes, fluoresceins, rhodamines, perylene dyes, croconium dyes, squarylium dyes, polymethine dyes, indocarbocyanine dyes, indodicarbocyanine dyes, indotricarbocyanine dyes, merocyanine dyes, phthalocyanines, naphthalocyanines, triphenylmethine dyes, croconium dyes, squarylium dyes, benzophenoxazine dyes, benzophenothiazine dyes, and derivatives thereof.

In an embodiment, a suited dye is selected from the group comprising polymethine dyes, indocarbocyanine dyes, indodicarbocyanine dyes, indotricarbocyanine dyes, merocyanine dyes, phthalocyanines, naphthalocyanines, triphenylmethine dyes, croconium dyes, squarylium dyes, and derivatives thereof.

In an embodiment, a suited dye is selected from the group comprising indocarbocyanine, indodicarbocyanine, indotricarbocyanine dyes and derivatives thereof. Examples are Cy7, Cy5.5, Cy3, AlexaFluor Dyes, indocyanine green (ICG).

In an embodiment, the diagnostic effector residue comprises a radioactive isotope. By such a radioactive isotope the polyglycerol derivative can be easily detected in the body of the human or the animal by standard methods of nuclear medicine.

In an embodiment, the diagnostic effector residue comprises an entity that can chelate a chemical substance, such as an atom, and ion or a molecule. In such a case, the diagnostic effector residue typically comprises said chelating entity (chelator) as well as a chelate bound fraction.

In an embodiment, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA) is used as chelator. Then, the polyglycerol derivative can be very well used in standard imaging methods being able to detect DOTA and the chelate bound fraction.

In an aspect, the instant invention relates to a method of site-specific imaging of angiogenesis, of extracellular matrix remodeling and/or of activated lymph nodes in a human or an animal in need thereof by detecting a polyglycerol derivative. Thereby, the polyglycerol derivative comprises on the one hand a dendritic polyglycerol backbone comprising a plurality of hydroxyl groups that are optionally at least partly substituted by sulfate groups and on the other hand a diagnostic effector residue. In an embodiment, the instant invention relates to method of diagnosing a disease at an early stage of the disease by applying the before-explained site-specific imaging.

All embodiments of the disclosed further medical use of a polyglycerol derivative can be combined in any desired way. In addition, all embodiments can be transferred to the explained methods, and vice versa.

The invention will be explained in more detail with respect to figures and exemplary embodiments. In the Figures:

Figure 1 A shows a sensorgram of the binding affinity of heparanase to dendritic polyglycerol sulfate (dPGS) ;

Figure 1 B shows a binding isotherm of the binding affinity of heparanase to dendritic polyglycerol sulfate (dPGS) ; and

Figure 2 shows an agarose electrophoresis gel depicting the inhibition of heparanase activity. Figure 1 A shows a surface plasmon resonance (SPR) sensorgram of the binding affinity of heparanase to dendritic polyglycerol sulfate (dPGS). Biotinylated dPGS was immobilized on a streptavidin-functionalized sensor chip to a final loading of 392 resonance units (RU). Two independent kinetic cycles with increasing amounts of heparanase (2.5, 7.4, 22.2, 66.7, and 200 nM) were run on a Biacore X1 00 device.

Figure 1 B depicts the binding affinity of heparanase to dendritic polyglycerol sulfate (dPGS) by a binding isotherm. By applying the steady state model, corresponding report points from the two sensorgrams shown in Figure 1 A were plotted and the KD value was calculated to be 92 nM. Thus, the binding of heparanase to surface-immobilized dendritic polyglycerol sulfate has been demonstrated ta have a KD value of 92 nM

Figure 2 shows the inhibition of heparanase activity, wherein fluorescently labeled heparin was used as a model substrate to monitor heparanase activity. 10 pmol heparin-FITC was incubated for 12 h at 37°C with 100 nM heparanase (lanes 2-6) and with decreasing amounts of dendritic polyglycerol sulfate (lane 3, 10 μΜ; lane 4, 1 μΜ; lane 5, 1 00 nM, and lane 6, 1 nM). Lane 1 is the untreated sample (only heparin-FITC) and lane 2 the heparanase-treated positive control (heparin-FITC plus heparanase). The samples were separated by agarose gel electrophoresis and visualized by fluorescence scanning.

From the results depicted in Figure 2, it can be clearly seen that dPGS provides protection from heparanase activity.