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The Developing Role of Peptide Radiopharmaceuticals in the Study of Chronic Inflammation: New Techniques for Novel Therapeutic Options

Posted on: Friday, 23 April 2004, 06:00 CDT

Chronic inflammatory diseases usually lead to fibrosis of the target organ and consequent hypo function. They are often relapsing, invalidating and require life-long treatment. In this class of patients it is very important to try and achieve specific immune suppression to extinguish the immune process with the aim of preventing the disease, preventing or delaying complications and avoiding disease relapse, often requiring surgical intervention. It is important that, while attempting to improve the quality of life of these patients by means of anti-inflammatory drugs, side effects are reduced to a minimum via the use of specific immune therapies that block as selectively as possible the pathologic mechanism responsible for the disease. New therapeutic options are being developed for specific targeted therapies. Several trials are being performed to assess the efficacy and safety of this approach. All of them, however, rely on the clinical assessment of the patients to evaluate the effect of treatment. It would be important to use an objective and reliable method to highlight directly the immune process underlying the individual disease. This manuscript reviews the radiopharmaceuticals available or recently developed for imaging chronic inflammatory diseases and their use for therapy decision making and follow-up.

KEY WORDS: Peptide - Radiopharmaceuticals - Cytokines Inflammation - Imaging radionuclide.

Chronic inflammatory diseases, such as graft rejection, sarcoiclosis, tuberculosis, autoimmune diseases and others, are characterized by long-standing mononuclear cell infiltration of the target organ eventually leading to organ malfunction. Atherosclerosis has also been recognized to be a chronic inflammatory disease.1

Nowadays, nuclear medicine techniques are not routinely used for the diagnosis of chronic inflammatory diseases but greatly contribute to the diagnosis and prognosis of the disease.2 Most importantly, nuclear medicine techniques are the most sensitive diagnostic modalities for the evaluation of the state of activity of the disease. This is of paramount importance since, in most cases, therapeutic options are available and prompt start of treatment may lead to disease prevention or delay of complications. The recent development of molecular nuclear medicine by the production of specific radiopharmaceuticals, moreover, further contributes to the identification of the immune process responsible for the individual dis ease. The referring physician, not only obtains information on the activity of the disease but also on the nature of the process and can, therefore, decide which treatment to start, when to start it and when to stop it or modify it.

In the following paragraphs a brief description of inflammatory processes will provide the rationale for the use of different radiopharmaceuticals in different diseases.

Pathophysiology of chronic inflammation processes

Acute inflammation lasts hours or days and is usually resolved with restitutio ad integrum; chronic inflammation can last from a few weeks to many years and usually causes late complications. There are several clinical and histological differences between acute and chronic inflammation that are mainly determined by the cause of the inflammation and its duration. Furthermore, 2 main types of chronic inflammation can be recognized: primary and secondary chronic inflammation.

Primary chronic inflammation

In this type of inflammation a distinctive chronic reaction is observed from the outset with little increased vascularity and permeability and little or no neutrophil infiltration. This is usually observed in cell-mediated immune responses against cells of the body that, following infection from intracellular microorganisms, generation of autoimmunity, cancer or transplantation, become the target of the immune response.3

Different cell types are present in tissue infiltrates in response to different inflammatory stimuli. Auto-immune diseases give rise to a whole spectrum of different histological characteristics ranging from a typical lymphocytic infiltration, as in thyroiclitis, to a mixed infiltrating cell population consisting of T and B cells, plasma cells and neutrophils, as in rheumatoid arthritis (RA), to a typically humoral mediated reaction, as in systemic lupus erythaematosus. Tumors also show infiltration by lymphomononuclear cells and in graft rejection a typical infiltration by cytotoxic lymphocytes is observed.4

Secondary chronic inflammation

This is due to the persistence of the stimulus that caused the acute inflammation. If an inflammatory agent persists, the character of the inflammatory lesion changes into chronic inflammation. Polymorpho-nuclear cells migrate out of the lesion; vasodilatation, vascular permeability and endothelial activation tend to normalize and the symptoms may disappear. The infiltrate becomes predominantly mononuclear, consisting of lymphocytes and cells of the monocyte- macrophage series, and further progression of the process is mainly via proliferation of infiltrating cells. Resolution of the process may take weeks or even years.4

Examples of this type of inflammation are chronic infections such as tuberculosis, leading to the formation of chronic granuloma, sarcoidosis and contact dermatitis.

Cell-mediated immunity and lymphocyte activation

Cellular immunity is based on specific antigen recognition and is mediated by T cytotoxic (CD8+ve) lymphocytes, macrophages and natural killer (NK) cells coordinated by T helper (CD4+ve) lymphocytes. Following the specific interaction of lymphocytes with the antigen and appropriate co-stimulatory signals, a complex series of phosphorylation reactions causes the transcription of genes that results in lymphocyte proliferation and differentiation. The cell starts synthesizing RNA and moves from GO into G1 phase of the mitotic cycle; proteins are synthesized, the cell enlarges and synthesis of interleukin-2 (IL2) and interleukin-2 receptor (IL2R) begins. Transferrin receptors are synthesized after 14 h, class II MHC molecules after 3-5 days and the very late activation antigen (VLA-4) after 7-14 days (Table I).4

Effects of inflammation on the endotbelium

The endothelium undergoes major changes that facilitate and enhance the recruitment of inflammatory cells and molecules. Permeability is increased to facilitate diffusion of macromolecules; adhesion molecules, such as ICAM-1, VCAM and E-selectins, are specifically expressed and bind to circulating granulocytes and lymphocytes promoting active migration into inflamed tissues. Somatostatin receptors have also been described on the endothelium of inflamed vessels.5,6 The targeting of inflamed vessels is a further option for the diagnosis of immune processes.

Effects of inflammation on the target tissue: apoplosis

Apoptosis is a term referring to the cytologically observable changes associated with a process of cellular self-destruction observed in all eukaryotes also called programmed cell death because it requires controlled gene expression which is activated in response to a variety of external or internal stimuli. This type of cell death involves cell activation through the engagement of cell surface receptors and subsequent signaling processes initiated from these receptors.

TABLE I.-Time course of lymphocyte activation in vitro.

During apoptosis cells shrink, develop bubble-like blebs on their surface, have the chromatin in their nucleus degraded, break into small, membrane-wrapped, fragments. One of the earliest events is the exposure on the surface membrane of the phospholipid phosphatidylserine, which is normally hidden within the plasma membrane. This is bound by receptors on phagocytic cells like macrophages and dendritic cells that engulf cell fragments.

Since intracellular contents are not released from apoptotic cells and their fragments, this process is not accompanied by inflammation and the process, therefore, can be regarded as an injury-limiting mode of cell disposal.

Apoptotic processes are physiologically observed during embryonal development, morphogenesis, metamorphosis, in endocrine tissue atrophy, during the normal turnover of tissues, and during tumor regression. Apoptosis also provides a defense mechanism against viruses by reducing virus spread through the rapid death of virus- infected cells and also eliminates cells with DNA damage.

Apoptotic cell death can be triggered by a variety of stimuli, including increased levels of oxidants within the cell, gamma irradiation, glucocorticoids or cytotoxic.7,8

APOPTOSIS AND INFLAMMATION

During inflammatory processes, cytotoxic T lympho-cytes secrete TNF-[alpha] and TNF-[beta] that, following binding to the TNF receptor, mediate apoptosis. Alternatively, "when cytotoxic T cells bind to their target, they produce more Fas ligand (FasL) at their surface. This binds with the Fas (also called CD95) on the surface of the target cell leading to its death by apoptosis. Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell. Apoptosis normally occurs during autoimmune diseases and organ transplantation and even in atherosclerotic plaques. Detection of apoptosis, therefore, in immune mediated disease holds important info\rmation on the ultimate effect of the inflammatory process on the target organ and can be very important to assess the efficacy of treatment.

Th1 and Th2 type of inflammation

Persistence of the antigen maintains the production of selected cytokines by immune cells that induce the differentiation of the effector cells responsible for the specific immune response. After the activation of T cells, their proliferation and maturation is regulated by the secretion of T cell soluble mediators, called cytokines, and by the expression of their specific receptors on the cell surface.

It has become clear that the type of antigen determines the type of immune response. Amongst T helper, CD4+ve, lymphocytes, 2 different subsets exist, called Th1 and Th2. They are responsible for maintaining 2 types of immune response by different cytokine production patterns; Th1 lymphocytes produce predominantly IL2, TNF[beta] and IFN[gamma] and are particularly effective in stimulating a cellular immune response whereas Th2 lymphocytes produce IL4, IL5, ILo, IL10 and are particularly effective in stimulating B lymphocytes and eosinophils. In particular it has been observed that IL4 can inhibit Th1 lymphocytes and IFN[gamma] Th2.

TABLE II.-Th1 or Th2 predominant response in human diseases.

It seems most likely that upon initial antigen stimulation T- helper lymphocytes secrete a mixture of Th1 and Th2 lymphokines and later differentiate, depending upon the nature of the antigenic stimulus. A high-affinity antigen peptide/TCR interaction would promote a Th1 response, whereas a low-affinity antigen peptide/TCR interaction would generate a Th2 response.9 The weak-affinity antigen/TCR interaction does not recruit CD45 and CD4 to the TCR complex and this may result in expression signaling molecules that promote Th2 and/or inhibit Th1 responses.

Recent in vivo studies also suggest that the anatomical route of primary allergen encounter (inhalation vs cutaneous) influenced the nature and the mechanism of the secondary Th2 response.10 Delivery of foreign antigens to mucosal surfaces, such as the pulmonary airways, has been shown to preferentially induce Th2-mediated immune responses in humans and in mice.

TH1 AND TH2 CELLS HAVE DIFFERENT CHEMOKINE RECEPTORS

Chemokines are cytokines that are chemotactic for (attract) leukocytes. Because they are chemotactic cytokines, chemokines are designated by the initials CC, Chemokines bind to receptors on the responding leukocyte. The receptors are transmembrane proteins with the chemokine binding site exposed at the surface of the plasma membrane. Chemokine receptors are designated CCR.

Th1 and Th2 lymphocytes have different functions and respond differently to chemokines through different membrane receptors: Th1 cells express the chemokine receptor CCR5 (but not CCR3) and Th2 cells express the chemokine receptor CCR3 (but not CCR5).11

CCR3 is found on Th2 cells, eosinophils and basophils, all cells implicated in allergic responses (.e.g., asthma). One chemokine that binds to CCR3 is called eotaxin. It is secreted by epithelial cells and phagocytic cells in regions where allergic reactions are occurring. CCR5 is found on Th1 cells and macrophages.

Th1 and Th2 network: clinical relevance and new opportunities for molecular nuclear medicine

It has become clear that different inflammatory dis eases are driven by a predominant Th1 or Th2 response (Table II). It is now possible, in an attempt to at more selective immune suppression, to start treatments that induce a shift from Th1 to Th2 or vice versa changing the inflammation from a pathogenic type to a non pathogenic one, in the individual disease. Th1-mediated diseases would benefit from therapies that induce a shift towards Th2 and vice versa.

Since Th1 and Th2 cells express distinct sets of chemokine receptors, it is envisaged that, by using radiolabelled ligands that specifically bind to the different Th subset would be able to identify in vivo the occurrence of the 2 types of inflammation.

With new therapeutic options, therefore, we are challenged not to verify whether the inflammation is active or not but to identify the type of inflammation. In this field, molecular nuclear medicine can offer unique information.

Novel therapeutic options

Chronic autoimmune disease, such as Crohn's disease, RA and others, are life-long and relapsing pathologies that require treatment with immuno-suppressive drugs (.e.g., corticosteroids, cyclosporin A, methotrexate) particularly in the more severe stages. Their efficacy, however, is limited and, in addition, year-or decade- long treatments are associated with significant toxicities.

Following increasing knowledge of the pathogenesis of the diseases, new specific therapies are becoming available for specific immuno-suppression, therefore achieving a more selective down regulation of auto reactive components that is usually obtained "with conventional anti-inflammatory steroid drugs.

This offers clinical benefit to autoimmune patients, that often require life-long treatment, while avoiding the unwanted toxic effects associated with high-dose or prolonged use of these compounds.

Shift from Th1 to Th2 inflammation in autoimmune diseases

STATINS

In a recent phase 1 clinical trial, a total of 45 patients with relapsing-remitting MS (RR-MS) received a daily oral dose of 80 mg of simvastatin for 6 months, and 41 were evaluated at the end of the study. Comparison of 3 monthly cranial MRI with 3 MRI performed at months 4, 5, and 6 following initiation of treatment showed a significant reduction (approximately 40%) in the mean number (P<0.0001) and in the mean volume (P<0.0016) of Gel-enhanced lesions (mean number pretreatment, 2.35 vs posttreatment, 1.31; mean volume pretreatment, 238 vs posttreatment, 142). No toxic effects were observed during the study.

A study on the use of atorvastatin in experimental autoimmune models of multiple sclerosis (MS) and in vitro experiments showed that oral administration atorvastatin prevented or attenuated chronic and relapsing paralysis in mice affected by demyelinating disease (experimental autoimmune encephalomyelitis); it also reduced cellular infiltrates in the central nervous system; blocked production of type 1 lymphokines (interleukin [IL]-2, IL-12, IFN- gamma and TNF alpha); promoted differentiation of CD4 cells toward a type 2 regulatory response; inhibited up regulation of MHC Class II antigens on microglia cells and of other co-stimulatory molecules involved in antigen presentation. Overall, atorvastatin blocked activation of immune cells, both by preincubation with antigen presenting cells or with T cells.12, 13

Thus, statins seem to be endowed with immune regulatory functions that go beyond the well-known effects on metabolic pathways.14

SELECTIVE INHIBITION OF IFN-GAMMA IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

The finding that a 50% reduction in the quantity of IFN-gamma produced was enough to prevent the disease in mice spontaneously developing SLE leads to hope that, even in humans, a partial reduction in IFN-gamma might be effective.15 This is important because it would allow selective manipulation of the immune system without generalized immunosuppression and potential preservation of other critical immune responses perhaps occurring at lower IFN- gamma thresholds, such as responses to infections and immune surveillance of cancer cell growth.

Theofilopoulos et al.15 thus designed a bivalent hybrid molecule consisting of the IFN-gamma receptor linked to the constant region of an immunoglobulin (IgG1 to confer a longer half-life in vivo, approximately 40 h). MRL/1pr mice injected prophylactically with a non viral vector expressing this IFN-gamma inhibitor did not develop the disease. Some beneficial effects were seen also when animals were treated at a more advanced disease stage.16

As noted by the authors, owing to the high number of processes influenced by IFN-gamma (e.g., antigen presentation, MHC expression on kidney cells, etc.) it is very difficult to pinpoint exactly the step, if any, affected by inhibition of the IFN-gamma system, that is critical for development of maintenance of lupus. Treatment was, however, associated with a reduction in the severity of the glomerulonephritis and in the deposition of immunocomplexes in the kidneys. The significant decrease in titre of polyclonal IgG and anti-chromatin IgG provides evidence that IFN-gamma plays a key role in breaking tolerance to chromatin.16

ORAL TOLERANCE

Inflammatory autoimmune diseases can be regarded as a failure of the immune system to maintain tolerance to certain self determinants. The treatment of these diseases is difficult since the aetiology is generally unknown and the immune response is directed to a wide range of self antigens in the target tissue. Oral tolerance (OT) consists of the oral administration of antigens (Ag) that could alter the response of the immune system. This is a form of peripheral immune tolerance in which mature lymphocytes in the peripheral lymphoid tissues are rendered non functional or hypo responsive by prior oral administration of Ag. This therapeutic approach requires the oral administration of Ag and the active participation of gut-associated lymphoid tissue (GALT), a tissue comprising Peyer's patches, intraepithelial cells and villi containing epithelials cells which is a well organized immune network. The mechanisms by which OT is mediated included deletion or anergy and active cellular suppression. The primary factor determining which form of tolerance will be developed after oral administration of Ag is Ag dosage.

Thus, it is thought that low doses of Ag induce the generation of active suppression. Active suppression is mediated in the GALT, precisely in the Peyer's patches, leading to the induction of regulatory T cells which then migrate to the systemic immune system and to the inflammatory area. One of the primary mechanism of active \cellular suppression is the secretion of down regulating or suppressive cytokines such as TGF[beta], IL4 and IL10 (17,18) that favours the induction of Th2 cells.

Conversely, a high dose of Ag favours clonal anergy or more rarely clonal deletion. High doses of antigen result in a systemic passage through the gut barrier, and a direct entrance to the systemic circulation, as intact protein that, in turn, contributes to determine unresponsiveness of Th1 cells via clonal anergy. A further mechanism by which OT operates is bystander suppression, the phenomenon in which regulatory cells, as generated by oral tolerance, are primed in an Ag specific manner, but act in the respective microenvironment in a non-Ag specific mariner. This phenomenon is of particular interest and explained the use of OT in T cell mediated autoimmune diseases such as RA, MS and type I diabetes, some diseases in which the auto-antigen remains unknown or where there are reactivities to multiple auto-antigens.

The ultimate effect of oral tolerance, therefore can be considered the inhibition of Th1 response by the stimulation of Th2 cytokines.

Therapeutic trials of oral tolerance in patients with rheumatoid arthritis

Th1 cells secreting TNF[alpha] and IFN[gamma] are found in joints of RA patients and thus may play an important role in the persistence of inflammation in RA synovia. OT which aimed to induce a shift in the Th1/Th2 balance seems promising and appropriate to RA treatment.19

There is a list of potential auto-Ag, the most popular being Type II collagen (CII) that is the most abundant protein of cartilage. In RA patients, B and T cell reactivity to CII has been demonstrated. Antibodies to CII have been detected in early and late stages of RA and the prevalence of such antibodies is known to range from 20% to 30%.

Based on these different aspects of RA pathophysiology and the mechanisms of action of OT, CII has been used in the treatment of RA patients.

Several studies have been performed in patients with different severity and duration of the disease. Over 500 patients were treated in different studies with a different amount of CII (20 to 2 500 g) for a variable period of time (3 to 6 months) while they were taken off their disease modifying anti-rheumatic (-DMARD) and immunosuppressive drugs. Results showed that 30% to 60% of patients showed a significant improvement in clinical indexes of disease activity (swollen and painful joint scores) in the CII treated group, In general low closes were more effective than high doses and patients with shorter duration and less severe degree showed the better response.20 Of interest, the presence of serum antibodies to CII at baseline was associated with the likelihood of responding to treatment.21 No side effects were recorded.

Shift from Th2 to Th1 in asthma

Th2 lymphocytes are considered to play an essential role in orchestrating the inflammatory response associated with asthma. In particular, Th2 lymphocytes express cytokines associated with IgE class switching and Th2 cell differentiation (IL4), eosinophil differentiation (interleukin-5), mucus secretion, airway smooth muscle hyperreactivity (IL13), and mast cell hyperplasia (IL9).

There have been a number of recent advances in our understanding of transcription factors, cytokines, and signal transduction molecules responsible for Th2 cell development and potential therapies to inhibit the Th2 response associated with asthma.

CpG motifs are noncoding sequences of DNA that are present in bacterial DNA and induce Th1 immune responses.22,23 In contrast to bacteria, humans have a much lower frequency of these CpG motifs and the CpG motifs in humans are methylated and nonfunctional. CpG motifs activate cells of the innate immune system (macrophages, NK cells) to generate cytokines such as interferon-gamma and IL12, which bias T cells to develop a Th1 as opposed to a Th2 immune response. The CpG motifs therefore do not directly act on T lymphocytes but act indirectly on T lymphocytes by inducing innate cell cytokines which bias the immune response to a Th1 response.

The potential applicability of CpG as a therapy for allergic disease was initially demonstrated in studies by Raz, from the University of California, San Diego, who demonstrated that CpG inhibits IgE responses in mice in vivo.24

Subsequent studies by other groups have demonstrated that CpG inhibits Th2 responses, eosinophilic inflammation and airway hyperreactivity in mouse models of asthma when administered either systemically or mucosally (intranasal or intratracheal).25 The inhibition of eosinophilic inflammation is mediated by an inhibition of the generation of IL5. CpG does not induce eosinophil apoptosis, but rather inhibits the generation of eosinophils in the bone marrow. Of interest, although corticosteroids are also able to inhibit IL5 and eosinophilic inflammation, unlike CpG they do not redirect the immune response to generate a Th1 immune response.

These studies in mice suggest that if CpG is effective in humans, the potential for CpG delivery as a metered-dose inhaler or an oral medication will need to be considered. Studies of CpG in human allergy and asthma are currently being performed and should shortly give us insight into whether CpG therapy is effective in humans as opposed to mouse models of allergy and asthma.

Peptide radiopharmaceuticals for the study of chronic inflammation

Radiolabelled peptides are a promising class of radiopharmaceuticals with diagnostic potential in several pathological conditions.

Peptides are small low molecular weight proteins (usually less than 10 000 Kd, less than 100 aminoacids). Many of them are natural peptides, some others are small "designer peptides", synthesized by DNA recombinant technology, constituted by a relatively small number of aminoacids (up to 30). In contrast to bigger proteins and antibodies, many peptides can be easily chemically synthesized, modified and stabilized to the metabolism to obtain optimized pharmacokinetic parameters.

Compared to larger molecules, like proteins and MoAbs, the biodistribution of peptides exhibits a rapid uptake and retention in target tissues with a good penetration into them, according with a usually rapid plasma clearance due to the renal excretion. This is particularly important for the study of chronic inflammation where hemodynamic changes are very poor and passive diffusion of macromolecules is very limited. Following intravenous injection, radiolabelled peptides accumulate in target tissues and organs via binding to specific receptors. They act as "probes" for the detection of target cells through external detection of body radioactivity by means of gamma-cameras, gamma-probes or PET scanners.

Several radiolabelled peptides have been used so far in oncology, neurology, metabolism and inflammation.26

Radiolabelled cytokines for imaging chronic inflammation

STRUCTURE AND FUNCTIONS OF CYTOKINES

Cytokines are proteins and glycoproteins members of a family of overlapping and interdependent molecules with important roles in the homeostatic control of the immune system and other organs, in physiology and pathology.27 Chemokines (CC, CXC, CX^sub 3^C, and C family) are chemotactic cytokines that control leukocyte trafficking both in physiological and inflammatory conditions, hematopoiesis, organogenesis and angiogenesis.28

Most cytokines are between 15 and 25 kD and they are often similar in size, charge and glycosylation.29 The major sources of cytokines are T cells and macro-phages but their production varies depending on the individual cytokine; IL2 is produced only by activated T cells whereas IL1 can be produced by macrophages, endothelial cells, large granular lymphocytes, T and B cells, fibroblasts, epithelial cells, astrocytes, keratinocytes and osteoblasts.

Cytokines act via the interaction with specific cell surface receptors expressed on a known cell population; cytokine receptors, usually of high affinity, are generally expressed at low levels on resting cells but their expression can be upregulated during activation.

PRINCIPLES OF THE USE OF RADIOLABELLED CYTOKINES IN DIAGNOSTIC IMAGING

The use of radiolabelled cytokines is a new field of molecular nuclear medicine. Their use allows us to study immune processes in vivo and, by contributing to the molecular characterization of diseased tissues, provides relevant information for the clinical management of patients.

As the pathophysiology underlying individual diseases differs, so does the cytokine network involved. Therefore, a cytokine suitable for the study of all pathological conditions does not exist. The optimal cytokine eligible for diseases- and stage-specific diagnosis will change according to the pathophysiological mechanisms operating in different diseases and to different clinical stages in the same disease.

In immune-mediated pathologies, radiolabelled cytokines contribute to differential diagnosis by confirming the immune nature of the disease, and to the study of the localization, severity and extent of the immune process. The major indication is the study of disease activity for the selection of patients eligible to treatment and for therapy follow-up.

The use of radiolabelled cytokines for diagnostic purposes is safe. Although several cytokines have unwanted biological effects at very low doses, radiolabelled cytokines are used in very small amounts (few micrograms) and generally do not induce side effects. They are human in origin and produced by recombinant DNA technology and are not immunogenic not even if used on repeated occasions for follow-up studies.30

Their use is easy and effective. Due to their low molecular weight they are rapidly cleared from plasma via the kidneys and easily concentrate into affected tissues infiltrated by inflammatory cells. Areas of pathologic uptake are easily differentiated from the background radioactivity of the body within a few hou\rs after the injection.31

Interleukin-2

IL2 is a single-chain .glycoprotein (MW: 15.5 kD) of 133 amino acids, synthesized and secreted, in vivo, by T lymphocytes after specific antigen stimulation.32

Its specific receptor is expressed on activated T cells. IL2 actions are: maintaining the immune response by inducing the long- term proliferation of activated T cells and the proliferation and differentiation of NK cells, B cells and macrophages.33 Infiltrating lymphocytes activate only after interaction with the antigen, consequently cells activation is an asynchronous process and follows a gradient of antigen concentration.

In inflammation foci activated lymphocytes express IL2R, becoming a target for radiolabelled IL2, while in physiological conditions the expression of IL2R in lymphoid tissues is negligible34,35 also in the course of inflammatory diseases peripheral blood cells do not express IL2R, consequently there is a very low binding of radiolabelled IL2 to circulating lymphocytes with low levels of background radioactivity.

The transient IL2R expression is an early lymphocyte activation marker.33 However, during the entire immune process, some of the lymphocytes activate and drives the inflammatory reaction, until the antigen is completely eliminated.

Radiolabelled IL2 allows the visualization of both lymphocytic infiltration and T lymphocyte activation. Evaluation of the state of activity of immune mediated processes is feasible through scintigraphy with this tracer. Interleukin-2 has been labeled with 35S, ^sup 125^I and tested in vitro.36,37 Biodistribution in rats has also been studied using ^sup 131^I-labelled IL-2.38

The reliability of ^sup 123^I-IL2 for the in vivo detection of lymphocytic infiltration, has been tested in animal models of human autoimmune diabetes, in Bio Breeding Worcester (BB/W) rats and in non obese diabetic (NOD) mice,39,40 and in rats with renal allograft.41 These studies in prediabetic animals have shown that there was uptake and retention of ^sup 123^I-IL2 in the pancreatic region between 5 and 15 minutes after injection. These data were confirmed by various experiments such as single organ counting after sacrifice of the animals, also autoradiography revealed that radioactivity was associated with infiltrating cells bearing IL2R. Histological examination of pancreata revealed a positive correlation between radioactivity and the degree of lymphocytic infiltration.

In man, studies in healthy volunteers and patients showed absence of significant biological effects at the low doses (2 to 30 g) used for imaging.42 Inflammatory bowel diseases,42,43 autoimmune thyroid diseases,44 insulin dependent diabetes,45 melanoma 46 and other different pathologies (Figure 1) have been investigated in over 400 patients.

Scintigraphy with ^sup 123^I-IL2, in patients with active Crohn's disease allowed the imaging of activated T lymphocytes. Since uptake by gut of ^sup 123^I-IL2 decreased after steroid therapy, its use has been hypothesized to select patients for immunosuppressive therapies and to monitor their efficacy.42 Microautoradiographies of specimens, demonstrated that ^sup 123^I-IL2 specifically bound to activated lymphocytes infiltrating the gut mucosa.

In coeliac patients the accumulation of ^sup 123^I-IL2 measured by gamma camera was consistent with the histological findings describing the number of infiltrating IL2R+ve cells in the jejunal mucosa.43

^sup 99m^Tc-IL2 accumulates in the thyroid of patients with Hashimoto's thyroiditis and Graves' disease. A study is being carried out to evaluate the reliability of ^sup 99m^Tc-IL2 as a marker for the prediction of relapse in Graves' disease.44

Scintigraphies with ^sup 99m^Tc-IL2 in newly diagnosed type 1 diabetic patients was performed at the time of clinical diagnosis. Results showed that ^sup 99m^Tc-IL2 accumulates at the time of diagnosis in the pancreas of patients with greater insulin production after 12 months treatment with nicotinamide. It may thus be inferred that IL2-scintigraphy identifies diabetic patients that at time of diagnosis have a persistent inflammation of residual beta- cell mass (insulitis) and may benefit from the use of immunomodulator drugs in preserving beta-cell function.47

In a recent comparative study between ^sup 99m^Tc-IL2 and ^sup 99m^Tc-HMPAO granulocytes in patients with inactive Crohn's disease it was shown that radiolabelled IL2 accumulated in most cases in areas that differed from that of radiolabelled granulocytes indicating that the 2 techniques detect different types of inflammation. Both techniques were characterized by high negative predictive value but ^sup 99m^Tc-IL2 was characterized by a better correlation with time to relapse.48

Currently the best approach to image chronic inflammation is the use of labelled IL2 although it binds to both Th1 and Th2 cells.

Octreotide

Somatostatin receptors are expressed on both activated lymphocytes and inflamed vascular endothelium. Somatostatin receptor scintigraphy (SRS) holds important information not only by demonstrating the presence of inflammation but also providing a rationale, in positive patients, for the use in selected cases, of unlabelled somatostatin for the treatment of the disease.

^sup 111^In-pentetreotide has been used in the imaging of Graves' disease, obtaining different contrasting results: a few studies have reported that this radiopharmaceutical accumulates in the thyroid and in the retro-orbital space in patients with exophthalmos and there is a positive correlation with the activity of disease, 49,50 in disagreement with other authors.44,51,52

One hypothesis has been formulated on mechanisms of accumulation of octreotide: uptake occurs in the early phases of Graves' ophthalmopathy when active infiltration is present; in the later stage of the disease, there is fibroblastic activity with subsequent fibrosis in the retro-orbital region without expression of somatostatin receptors.53,54 It is not suitable for the imaging of experimental abscesses.55

Figure 1.-^sup 99m^Tc-IL2 scintigraphy in a patient with active scleroderma. Accumulation of radiolabelled IL2 is seen at both forearms of the patients (top) whereas no accumulation is seen in a control subject (bottom).

SSRs were observed in vitro in multiple confluent granulomas in patients with active sarcoidosis, the receptors are not on the surface of lymphocytes but are located in the areas containing epithelial cells. In patients successfully treated with steroids with complete sclerosis of the granulomatous lesion SSRs were not found. These studies are in agreement with studies in patients with tuberculosis.56 A recent study by Lebtahi et al. compared the use of ^sup 111^In-pentetreotide to that of ^sup 67^Ga-citrate in patients with sarcoidosis, showing similar diagnostic accuracy.57

Hyper-expression of the somatostatin receptor (SSR) has been found in intestinal samples from patients with active ulcerative colitis and Crohn's disease. SSRs were localized in intramural veins and were not detected in non-inflamed control intestine.58 SSRs were reported in vitro in patients with active RA.59

Figure 2.-^sup 99m^Tc-HYNIC-EDDA-TOC scintigraphy in a patient with active rheumatoid arthritis. Accumulation of radiolabelled ^sup 99m^Tc-HYNIC-EDDA-TOC is observed at both knees indicating active inflammation of the synovium.

This radiopharmaceutical is applicabile in the imaging of chronic inflammation, but is unsuitable for visualization of acute infectious disease.55

A study was performed with ^sup 111^In-pentetreotide orbital scintigraphy on patients with severe ophthalmopathy caused by Graves' disease, Hashimoto's thyroiditis, Means' Syndrome. Activated lymphocytes express SSR during the active phase of the disease, permitting ^sup 111^In-pentetreotide scintigraphy.

The authors concluded that ^sup 111^In-pentetreotide scintigraphy makes it possible to select patients for octreotide therapy, which seems to be adequate in active, moderately severe thyroid eye disease, especially when it involves soft tissues.60-63

A recent study has described the use of a new somatostatin analogue ^sup 99m^Tc-HYNIC/EDDA-tyr(3)-octreotide (HYNIC/EDDA-TOC) for the diagnosis of the state of activity in patients with Sjogren syndrome. Results showed that inflamed parotid glands could be diagnosed by this radiopharmaceutical. Inflamed joints were also detected in patients with active RA (Figure 2).64

Substance P

Substance P is a neuroendocrine peptide, whose receptor has been found hyper-expressed on the endothelial cells of vessels in inflammatory sites,65 in patients with RA,66 in vessels in surgical specimens from patients with ulcerative colitis and Crohn's disease. ^sup 111^In-Substance P has been tested in the detection of inflammation in animal models,67 and ^sup 111^In-Substance P has been employed in imaging of the thymus and inflammatory foci in immunomediated disease, with a higher uptake than ^sup 111^In- octreotide.68 Unfortunately cardiovascular side effects were found 69 such as transient flush and in 1 patient significant hypotension associated with bradycardia, the development of an antagonist in the future might therefore be a clinical application.

Tuftsin antagonists

Tuftins is a chemotactic tetrapepticle (TKPR) derived from the Fc portion of IgG, and promotes chemotaxis and phagocytosis of neutrophils, macrophages and monocytes by a receptor-mediated mechanism. An antagonist (TKPPR) has been studied by Goodbody et al.70 In rats with inflammatory bowel disease, infection-to- background ratios using ^sup 99m^Tc-labelled tuftsin antagonist were higher than using ^sup 111^In-labelled leukocytes. Experiments with ^sup 99m^Tc-labelled tuftsin antagonist, in rats with E. Coli infections, showed an increase of target-to-background ratios up to 16 at 17 h post injection. However the following disadvantages have been found: absolute uptake in the infection was \low, there was uptake by kidneys and liver.71 Rapid whole-body clearance was found by scintigraphy in healthy volunteers, and accumulation by synovium of the major joints was also found.72 The pepticle does not yield side-effects, it is well tolerated and has been tested on patients with RA. Another analogue, RMT-1 has been studied in various animals affected by sterile inflammation and infection.73

In a pilot phase 2 clinical study ^sup 99m^Tc-RP128 has made it possible to detect inflammatory lesions in patients with Chrohn's disease within 4 h postinjection.74, 75

Recently, a bifunctional peptide chelate, ^sup 99m^Tc-RP128 has been designed as a tuftsin receptor antagonist. RP128 consists of a tripeptide N3S ^sup 99m^Tc-chelator, dimethyl-Gly-Ser-Cys (acetamidomethyl), linked by a glycine residue to the targeting domain composed of the tuftsin antagonist TKPPR. This pentapeptide analog has a fourfold greater receptor affinity than tuftsin.76 A study has investigated the safety, tolerance, normal biodistribution and dosimetry of this compound in healthy volunteers. In patients with active RA a markedly increased uptake by several affected joints has been observed. ^sup 99m^Tc-RP128 was cleared rapidly from the blood by renal excretion, it did not accumulate in major organs. Then, ^sup 99m^Tc-RP128 provided images of inflammation foci rapidly and with high target-to-background ratios.77

The potential of ^sup 99m^Tc-RP128 to image central nervous system (CNS) inflammation in experimental allergic encephalomyelitis (EAE) was evaluated in an animal model of MS. There was remarkable uptake in the inflamed sites at 0.5, 1 and 3 hours post injection. Since this uptake decreased with a dose-response pattern after glucocorticoid therapy, the tracer is reliable for assessing the efficacy of the therapy.78

Imaging of apoptosis

Radiolabelled Annexin-V, an endogenous human protein, binding with high affinity phosphatidilserine, makes it possible to assess non invasively for the early stage of apoptosis, before DNA degradation and membrane vescicle formation, providing information about disease progression or regression and about the efficacy of a therapy for inhibition or induction of cell death. This technique is not able to distinguish between apoptosis and necrosis, because phosphatidilserine is externalized during both processes, but the information acquired is of fundamental importance in clinical practice.

A recent study has tested the reliability of ^sup 99m^Tc-HYNIC- Annexin-V in the study of acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts, in fulminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice, in cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. In control animals the radiotracer visualized only the kidney, whereas in mice affected by fulminant hepatitis the liver was clearly visualized at 1 h, with increase of uptake up to 2 h, with concomitant decrease of renal activity. The radiopharmaceutical also provided clear images in the PVG cardiac allografts, scintillation well-counting assay showed an 11-fold increased uptake in PVG allograft as compared with native heart activity. Scintillation camera imaging revealed a decrease of uptake in treated flank tumors with respect to untreated flank tumor implants, in agreement with results obtained with scintillation well- counting assay. All these data were confirmed by immunostaining assay.79

The usefulness of ^sup 99m^Tc-HYNIC-Annexin-V to image apoptotic processes in vivo has been evaluated in mice treated with dexamethasone to induce thymic apoptosis, in Fas-defective mice (lrp/ lrp) in which administration of anti-Fas antibody does not induce hepatic apoptosis, in wild-type mice treated with anti-Fas antibody, to produce massive apoptosis similar to fulminant hepatitis. The Fas/ Fas ligand system modulates the elimination of lymphocyte clones in the immune system, indeed, an excessive proliferation of these cells promotes immunologic pathologies similar to the syndrome of lupus erythematosus and RA. Biodistribution studies have revealed uptake by kidney, and a rapid blood clearance (after 30 min there was less radioactivity than 23% of the injected dose in the blood), a minimal uptake by bone marrow. In mice treated "with dexamethasone, a lympholytic agent, a remarkable increase in thymic uptake was observed, in agreement with data obtained with fluorescent cell sorting analysis. lrp/lr pmice are genetically deficient in Fas receptor expression, actually scintigraphy has not imaged any uptake after administration of anti-Fas antibody Jo-2. Indeed in wild-type mice treated with anti-Fas antibody there was a considerable increase in tracer uptake by the liver. All these results were confirmed by histologic analysis and by TUNEL staining of hepatocytic nuclei.80

The use of ^sup 99m^Tc-HYNIC-Annexin-V has been described in monitoring therapies in acute myocardial infarction.81

A recent study described the use of ^sup 99m^Tc-Annexin-V in an experimental model of RA. Results showed that radiolabelled Annexin- V accumulated with high target/background values in affected joints. Annexin-V uptake significantly decreased after steroid treatment.82

Future perspectives: peptide ligands for the specific detection of Th1/Th2 inflammation

Interleukin-12

IL12 is secreted by peripheral lymphocytes after activation. IL12 is a heterodimeric 70 kDa glycoprotein (IL12-p70 ) consisting of a 40 kDa subunit and a 35 kDa subunit linked by disulficle bonds that are essential for the biological activity of IL12. The IL12 receptor (CD212) appears to be a single protein of approximately 110 kDa. Up to 1 000-9 000 high affinity IL12 receptors/cell are expressed on peripheral blood mononuclear cells activated by various T-cell mitogens or by IL2. IL12 receptors are present on activated T-cells expressing CD4 and CDS and on activated CD56 positive natural killer cells. IL12 stimulates the proliferation of human lymphoblasts following cell activation by phytohemagglutinin. IL12 activates NK- cells positive for CD56, and this activity is blocked by antibodies specific for TNF-alpha. In peripheral lymphocytes of the ThI T- helper cell type IL12 induces the synthesis of IFN-gamma, IL2 and TNF, promotes and enhances Th1 function and differentiation and inhibits Th2 lymphocytes.83

FIRST EXPERIENCE WITH RADIOLABELLED INTERLEUKIN-12P40

The IL12-p40 subunit lacks any biological activity, and retains the ability to bind the IL12 receptor, although with lower affinity than the heterodimeric cytokine. This property makes IL12 p40 suitable candidate for in vivo imaging of Th1 cells without the risk of inducing side-effects.

IL12-p40 was labeled with ^sup 125^I using the enzymatic method (LPO-GOD) and purified by gel filtration chromatography. Labeling efficiency ranged between 58-79%. Scatchard Plot analysis on Kit- 225 cells has ascertained the retention in vitro, of the affinity of labeled IL12-p40 for its receptors. Biodistribution studies in animals have assessed the hepatic metabolism of radiolabeled IL12- p40 and its uptake in sites where the Kit-225 cells (a Th1 cell line that express the IL12 receptor) were experimentally injected.84

This radiolabelled cytokine seems useful for the in vivo imaging of Th1-mediated inflammatory processes. Human biodistribution is being studied.

RADIOLABELLED IL12

A recent study by Annovazzi et al. tested the capability of ^sup 123^I-IL12 to image in vivo experimentally induced Th1 colitis in mice. IL12 was labeled to high specific activity with retained receptor binding capacity. Results showed that radiolabelled IL12 only accumulates in affected animals compared to controls.85

Eotaxin

This protein of 73 amino acids has been found in the bronchoalveolar lavage of guinea pigs used as a model of allergic inflammation. Eotaxin is also referred to as Eotaxin-1 (following the identification of related factors Eotaxin-2 and Eotaxin-3) and has been renamed CCL11. Eotaxin induces substantial eosinophil accumulation at a 1-2 pmol close in the skin without significantly affecting the accumulation of neutrophils. Recombinant human eotaxin induces a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response cannot be desensitized by pretreatment of eosinophils with other CC-Chemokines, suggesting a unique receptor. The receptor (CCR3) is a G-protein-coupled receptor selectively expressed in human eosinophils.86, 87 The characteristics of eotaxin make it a potential candidate for the selective in vivo detection of Th2 response. At present, however, no studies are available in vivo with radiolabelled eotaxin.

Conclusions

The progress of our understanding and knowledge of the pathophysiology of chronic inflammatory dis eases has prompted the development of several new specific receptor-binding peptide radiopharmaceuticals. Data obtained so far on the clinical use of radio-labelled peptides have demonstrated that this approach is safe and effective in vivo for the study of selected cell populations in several inflammatory conditions. It is now possible to assess the activity of the disease, but also to verify its ultimate effects: cell killing. New "tailored peptides" are being continuously tested in vivo to match new diagnostic challenges. It is hoped that by newer specific approaches nuclear medicine will increasingly contribute to the clinical management of patients with inflammatory diseases.

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