2 Pathophysiology and Mechanisms of Radiopharmaceutical Localization

2 Pathophysiology and Mechanisms of

Radiopharmaceutical Localization

Shankar Vallabhajosula, Azu Owunwanne

2.1 Definition of Disease 29 2.2 Pathophysiology 29

2.3 Altered Cellular and Tissue Biology 30 2.3.1 Cellular Adaptations 30

2.3.2 Cellular Injury 30 2.3.3 Necrosis 31 2.3.4 Apoptosis 31

2.4 Radiopharmaceuticals 32

2.5 Mechanism(s) of Radiopharmaceutical Localization 34

2.5.1 Isotope Dilution 35 2.5.2 Capillary Blockade 35

2.5.3 Physicochemical Adsorption 35 2.5.4 Cellular Migration and Sequestration 35 2.5.5 Membrane Transport 36

2.5.5.1 Simple Diffusion 36 2.5.5.2 Facilitated Diffusion 38 2.5.5.3 Active Transport 38 2.5.5.4 Phagocytosis 39

2.5.5.5 Receptor-Mediated Endocytosis 40 2.5.6 Metabolic Substrates and Precursors 40 2.5.6.1 Precursors: Radiolabeled Amino Acids 40 2.5.7 Tissue Hypoxia 41

2.5.8 Cell Proliferation 41 2.5.9 Specific Receptor Binding 42 2.5.9.1 Radiolabeled Peptides 42 2.5.9.2 Steroid Hormone Receptors 43

2.5.9.3 Adrenergic Presynaptic Receptors and Storage 44 2.5.9.4 LDL Receptors 44

2.5.9.5 Radiolabeled Antibodies 44 2.5.10 Imaging Gene Expression 46 References 47

2.1

Definition of Disease

At the present time, the precise definition of disease is as complex as an exact definition of life. It may be rela- tively more easy to define disease at a cellular and mo- lecular level than at the level of an individual. Through- out the history of medicine, two main concepts of dis- ease have predominated [1]. The ontological concept views a disease as an entity that is independent, self- sufficient, and runs a regular course with a natural his- tory of its own. The physiological concept defines dis- ease as a deviation from normal physiology or bio- chemistry; the disease is a statistically defined devia-

tion of one or more functions from those of healthy people under circumstances as close as possible to those of a person of the same sex and age of the patient.

The term homeostasis is used by physiologists to mean maintenance of static, or constant, conditions in the internal environment by means of positive and nega- tive feedback of information. About 56% of the adult hu- man body is fluid. Most of the fluid is intracellular, and about one third is extracellular fluid that is in constant motion throughout the body and contains the ions (sodi- um, chloride, and bicarbonate) and nutrients (oxygen, glucose, fatty acids, and amino acids) needed by the cells to maintain life. Claude Bernard (1813 – 1878) described extracellular fluid as the internal environment of the body and hypothesized that the same biological process- es that make life possible are also involved in disease [1].

The laws of disease are the same as the laws of life [1]. As long as all the organs and tissues of the body perform functions that help to maintain homeostasis, the cells of the body continue to live and function properly.

At birth, molecular blueprints collectively make up a person’s genome or genotype that will be translated into cellular structure and function. A single gene defect can lead to biochemical abnormalities that produce many dif- ferent clinical manifestations of disease, or phenotypes, a process called pleotropism. Many different gene abnor- malities can result in the same clinical manifestations of disease – a process called genetic heterogeneity. Thus, diseases can be defined as abnormal processes as well as abnormalities in molecular concentrations of different biological markers, signaling molecules, and receptors.

2.2

Pathophysiology

In the year 1839, Theodor Schwann discovered that all living organisms are made up of discrete cells [2]. In 1858, Rudolph Virchow observed that a disease could not be understood unless it were realized that the ulti- mate abnormality must lie in the cell. He correlated dis- ease with cellular abnormalities as revealed by chemical stains, thereby founding the field of cellular pathology.

He defined pathology as physiology with obstacles [2].

Chapter 2

Most diseases begin with cell injury, which occurs if the cell is unable to maintain homeostasis. Since the time of Virchow, gross pathology and histopathology have been a foundation of the diagnostic process and the classification of disease. Traditionally, the four aspects of a disease process that form the core of pathology are etiology, pathogenesis, morphological changes, and clinical significance [3]. The altered cellular and tissue biology and all forms of loss of function of tissues and organs are ultimately the result of cell injury and cell death. Therefore, knowledge of the structural and func- tional reactions of cells and tissues to injurious agents, including genetic defects, is the key to understanding the disease process. Currently, diseases are defined and interpreted in molecular terms and not just as general descriptions of altered structure. Pathology is evolving into a bridging discipline that involves both basic sci- ence and clinical practice and is devoted to the study of the structural and functional changes in cells, tissues, and organs that underlie disease [3]. The use of molecu- lar, genetic, microbiological, immunological, and mor- phological techniques is helping us to understand both ontological and physiological causes of disease.

2.3

Altered Cellular and Tissue Biology

The normal cell is able to handle normal physiological and functional demands, so-called normal homeostasis.

However, physiological and morphological cellular ad- aptations normally occur in response to excessive physi- ological conditions or to some adverse or pathological stimuli [3]. The cells adapt in order to escape and protect themselves from injury. An adapted cell is neither nor- mal nor injured but has an altered steady state, and its viability is preserved. If a cell cannot adapt to severe stress or pathological stimuli, the consequence may be cellular injury that disrupts cell structures or deprives the cell of oxygen and nutrients. Cell injury is reversible up to a certain point, but irreversible (lethal) cell injury ultimately leads to cell death, generally known as necro- sis. By contrast, an internally controlled suicide pro- gram, resulting in cell death, is called apoptosis.

2.3.1

Cellular Adaptations

Some of the most significant physiological and patho- logical adaptations of cells involve changes in cellular size, growth, or differentiation [3, 4]. These include (a) atrophy, a decrease in size and function of the cell; (b) hypertrophy, an increase in cell size; (c) hyperplasia, an increase in cell number; and (d) metaplasia, an alter- ation of cell differentiation. The adaptive response may also include the intracellular accumulation of normal

endogenous substances (lipids, protein, glycogen, bili- rubin, and pigments) or abnormal exogenous products.

Cellular adaptations are a common and central part of many disease states. The molecular mechanisms leading to cellular adaptation may involve a wide variety of stim- uli and various steps in cellular metabolism. Increased production of cell signaling molecules, alterations in the expression of cell surface receptors, and overexpression of intracellular proteins are typical examples.

2.3.2

Cellular Injury

Cellular injury occurs if the cell is unable to maintain homeostasis. The causes of cellular injury may be hyp- oxia (oxygen deprivation), infection, or exposure to toxic chemicals. In addition, immunological reactions, genetic derangements, and nutritional imbalances may also cause cellular injury. In hypoxia, glycolytic energy production may continue, but ischemia (loss of blood supply) compromises the availability of metabolic sub- strates and may injure tissues faster than hypoxia. Vari- ous types of cellular injury and their responses are summarized in Table 2.1.

Biochemical Mechanisms. Regardless of the nature of injurious agents, there are a number of common bio- chemical themes or mechanisms responsible for cell in- jury [4].

1. ATP depletion: Depletion of ATP is one of the most common consequences of ischemic and toxic inju-

Table 2.1. Progressive types of cell injury and responses (from [3])

Type Responses

Adaptation Atrophy, hypertrophy, hyperplasia, metaplasia

Active cell injury Immediate response of “entire cell”

Reversible Loss of ATP, cellular swelling, detach- ment of ribosomes, autophagy of lyso- somes

Irreversible “Point of no return” structurally when vacuolization of the mitochondria oc- curs and calcium moves into the cell Necrosis Common type of cell death with severe

cell swelling and breakdown of organ- elles

Apoptosis Cellular self-destruction to eliminate unwanted cell population

Chronic cell inju- ry (subcellular alterations)

Persistent stimuli response may involve only specific organelles or cytoskeleton, e.g., phagocytosis of bacteria

Accumulations or infiltrations

Water, pigments, lipids, glycogen, pro- teins

Pathological calcification

Dystrophic and metastatic calcification

ry. ATP depletion induces cell swelling, decreases protein synthesis, decreases membrane transport, and increases membrane permeability.

2. Oxygen and oxygen-derived free radicals: Ischemia causes cell injury by reducing blood supply and cellular oxygen. Radiation, chemicals, and inflam- mation generate oxygen free radicals that cause de- struction of the cell membrane and cell structure.

3. Intracellular Ca

and loss of calcium homeostasis:

Most intracellular calcium is in mitochondria and endoplasmic reticulum. Ischemia and certain toxins increase the concentration of Ca

in cyto- plasm, which activates a number of enzymes and causes intracellular damage and increases mem- brane permeability.

4. Mitochondrial dysfunction: A variety of stimuli (free Ca

levels in cytosol, oxidative stress) cause mitochondrial permeability transition (MPT) in the inner mitochondrial membrane, resulting in the leakage of cytochrome c into the cytoplasm.

5. Defects in membrane permeability: All forms of cell injury and many bacterial toxins and viral pro- teins damage the plasma membrane. The result is an early loss of selective membrane permeability.

Intracellular Accumulations. Normal cells generally accumulate certain substances such as electrolytes, li- pids, glycogen, proteins, calcium, uric acid, and biliru- bin that are involved in normal metabolic processes. As a manifestation of injury and metabolic derangements in cells, abnormal amounts of various substances, ei- ther normal cellular constituents or exogenous sub- stances, may accumulate within the cytoplasm or in the nucleus, either transiently or permanently. One of the major consequences of failure of transport mecha- nisms is cell swelling due to excess intracellular fluid.

Abnormal accumulations of organic substances such as triglycerides, cholesterol and cholesterol esters, glyco- gen, proteins, pigments, and melanin may be caused by disorders in which the cellular capacity exceeds the synthesis or catabolism of these substances. Dystrophic calcification occurs mainly in injured or dead cells, while metastatic calcification may occur in normal tis- sues due to hypercalcemia that may be a consequence of increased parathyroid hormone, destruction of bone tissue, renal failure, and vitamin-D-related disorders.

All these accumulations harm cells by “crowding” the organelles and by causing excessive and harmful me- tabolites that may be retained within the cell or ex- pelled into extracellular fluid and circulation.

2.3.3 Necrosis

Necrosis is cellular death resulting from the progressive degradative action of enzymes on the lethally injured

cells, ultimately leading to the processes of cellular swelling, dissolution, and rupture. The morphological appearance of necrosis is the result of denaturation of proteins and enzymatic digestion (autolysis or hetero- lysis) of the cell. Different types of necrosis occur in dif- ferent organs or tissues. The most common type is coa- gulative necrosis, resulting from hypoxia and ischemia.

It is characterized by denaturation of cytoplasmic pro- teins, breakdown of organelles, and cell swelling, and it occurs primarily in the kidneys, heart, and adrenal glands. Liquefactive necrosis may result from ischemia or bacterial infections. The cells are digested by hydro- lases and the tissue becomes soft and liquefies. As a re- sult of ischemia, the brain tissue liquefies and forms cysts. In infected tissue, hydrolases are released from the lysosomes of neutrophils; they kill bacterial cells and the surrounding tissue cells, resulting in the accu- mulation of pus. Caseous necrosis, present in the foci of tuberculous infection, is a combination of coagulative and liquefactive necrosis. In fat necrosis, the lipase en- zymes break down triglycerides and form opaque, chalky necrotic tissue as a result of saponification of free fatty acids with alkali metal ions. The necrotic tis- sue and the debris usually disappear by a combined process of enzymatic digestion and fragmentation or they become calcified.

2.3.4 Apoptosis

Apoptosis, a type of cell death implicated in both nor- mal and pathological tissue, is designed to eliminate unwanted host cells in an active process of cellular self- destruction effected by a dedicated set of gene prod- ucts. Apoptosis occurs during normal embryonic de- velopment and is a homeostatic mechanism to main- tain cell populations in tissues. It also occurs as a de- fense mechanism in immune reactions and during cell damage by disease or noxious agents. Various kinds of stimuli may activate apoptosis. These include injurious agents (radiation, toxins, free radicals), specific death signals (TNF and Fas ligands), and withdrawal of growth factors and hormones. Within the cytoplasm a number of protein regulators (Bcl-2 family of proteins) either promote or inhibit cell death. In the final phase, the execution caspases activate the proteolytic cascade that eventually leads to intracellular degradation, frag- mentation of nuclear chromatin, and breakdown of cy- toskeleton. The most important morphological charac- teristics are cell shrinkage, chromatin condensation, and the formation of cytoplasmic blebs and apoptotic bodies that are subsequently phagocytosed by adjacent healthy cells and macrophages. Unlike necrosis, apo- ptosis is nuclear and cytoplasmic shrinkage and affects scattered single cells.

2.3 Altered Cellular and Tissue Biology

31

2.4

Radiopharmaceuticals

Chemistry is the language of health and disease, be- cause the body is a vast network of interacting mole- cules. If the definition of the disease is molecular, diag- nosis becomes molecular [5]. Because the treatment of many diseases is chemical, it becomes more and more appropriate that chemistry be the basis of diagnosis and of the planning and monitoring of treatment [1].

Nuclear medicine, in the simplest terms, is the medical specialty based on examining the regional chemistry of the living human body. In the 1920s, Georg DeHevesy coined the term “radioindicator” (radiotracer) and in- troduced the “tracer principle” to the biomedical sci- ences [1]. One of the most important characteristics of a true tracer is the ability to study the components of a homeostatic system without disturbing their function.

Since the physiological approach defines a disease in terms of the failure of a normal physiological or bio- chemical process, the nuclear medicine diagnostic pro- cedures involve four types of measurement: (a) region- al blood flow, transport, and cellular localization of var- ious molecules; (b) metabolism and bioenergetics of

Table 2.2. Radiopharmaceuticals for diagnostic imaging studies

Radiopharmaceutical Application Indication for imaging

Radiolabeled particles

99mTc-MAA, 10 – 50 µm Capillary blockade Lung perfusion

99mTc-DTPA, aerosol, 1 – 4 µm Sedimentation in bronchioles Lung ventilation

99mTc-Sulfur colloid, 0.1 – 1.0 µm Reticuloendothelial function Liver, spleen, and bone marrow

99mTc-SC, filtered 0.1 – 0.3 µm Lymphatic drainage Breast cancer and melanoma

99mTc-HSA (nanocolloid), 0.02 µm Lymphatic drainage Breast cancer and melanoma

99mTc-Antimony sulfide colloid, 0.1 µm Lymphatic drainage Breast cancer and melanoma Radiolabeled gases

133Xe,¹²⁷Xe,^81mKr Alveolar transit-capillary diffusion Lung ventilation

99mTc-Technegas, 0.004 – 0.25 µ Alveolar transit-capillary diffusion Lung ventilation Radiolabeled chelates

99mTc-MDP, HDP Bone formation Metastatic bone disease, neuroblastoma,

osteosarcoma

99mTc-DTPA Blood brain barrier disruption Brain tumors

Renal function glomerular filtration Renal blood flow and renogram

99mTc-MAG3 Renal function, tubular secretion Renogram

99mTcIII-DMSA Binding to renal parenchyma Renal scan

99mTcV-DMSA Tumor cell uptake Medullary carcinoma of thyroid

99mTc-Disofenin and mebrofenin Hepatobiliary function Hepatobiliary imaging

99mTc-Ceretec and Neurolite Blood flow Brain imaging

99mTc-sestamibi and tetrafosmin Blood flow Myocardial perfusion

99mTc-sestamibi, and tetrafosmin Tumor viability and multidrug resistance, MDR (Pgp expression)

Breast cancer, parathyroid adenoma, brain tumor

111In-DTPA CSF flow Cisternogram

111In-oxine Radiolabeling white cells Labeled leukocyte thrombus imaging

67Ga-citrate Tumor viability, capillary leakage Tumor and infection imaging Radiotracers as ions

99mTc-pertechnetate (TcO4–) Thyroid function (trapping) Thyroid imaging

123I,¹³¹I-sodium iodide (I^–) Thyroid function (trapping) Thyroid uptake, imaging therapy

82Rb-chloride, Rb⁺ Blood flow Myocardial perfusion

201Tl-thallous chloride, Tl(OH)2+ Blood flow Myocardial perfusion

Tumor viability Tumor imaging (brain, parathyroid, thyroid)

tissues; (c) physiological function of organs; and (d) in- tracellular and intercellular communication.

A number of radiopharmaceuticals (Table 2.2) have been designed and developed over the past four de- cades to image the structure and function of many or- gans and tissues. Radiopharmaceutical agents exhibit a huge range of physical and chemical properties and may be classified into eight different categories. The most important factors that influence the transport, uptake, and retention of radiopharmaceuticals in dif- ferent organs and tissues include the chemical and bio- chemical nature of the carrier molecule transporting the radionuclide of choice to the targeted area. The use of radiopharmaceuticals to deliver therapeutic doses of ionizing radiation has been extensively investigated.

Targeted radionuclide therapy by systemic administra-

tion of a radiopharmaceutical provides a potential to

treat widely disseminated cancer tissue. A number of

radiopharmaceuticals (Table 2.3) are now available for

the treatment of different malignancies or palliation of

pain due to bony metastases. Tumor-specific radio-

pharmaceuticals that are clinically useful for noninva-

sive imaging of tumors are being modified for radionu-

clide therapy of tumors.

Table 2.2. (cont.)

Radiopharmaceutical Application Indication for imaging

Radiolabeled cells

111In-leukocytes Cell migration and phagocytosis Infection imaging

111In-platelets Cell incorporation in thrombus Thrombus imaging

51Cr-RBCs Dilution in blood compartment RBC mass and blood volume

99mTc-RBCs Cardiac function Cardiac ejection fraction, wall motion

Blood pool Hemangioma, GI bleeding

99mTc-RBC (heat denatured) Spleen Accessory splenic tissue

Receptor binding radiotracers

111In-pentetreotide, Octreoscan Somatostatin receptors Neuroendocrine tumors

99mTc-P829, Neotec Somatostatin receptors Lung cancer, NE tumors

99mTc-P280, Acutect GP IIb/IIIa receptors Thrombus imaging, DVT

99mTc-TRODAT-1 Dopamine transporter Brain imaging-dopamine D2 receptors

123I-VIP VIP receptors Gastrointestinal tumors

131I-NP-59 LDL receptor, cholesterol metabolism Adrenal carcinoma, adenoma, Cushing’s syndrome

123I- or¹³¹I-MIBG Presynaptic adrenergic receptors Myocardial failure

Adrenergic tissue uptake Tumor imaging (pheochromocytoma, neuroendocrine, neuroblastomas) [¹¹C]Raclopride Dopamine D2 receptors Brain imaging-dopamine D2 receptors

123I-IBZM Dopamine D2 receptors Brain imaging-dopamine D2 receptors,

tumor imaging, malignant melanoma [¹⁸F]fluoro-estradiol (FES) Estrogen receptors Breast tumor imaging

Radiolabeled monoclonal antibodies

111In-Oncoscint, B72.3 IgG TAG-72 antigen Colorectal and ovarian cancer

111In-Prostascint, 7E11-C5.3 IgG PSMA (intracellular epitope) Prostate cancer

99mTc-CEA-Scan, IMMU-4 Fab’ CEA Colorectal cancer

99mTc-Verluma, NR-LU-10 Fab’ Cell surface GP as antigen Small cell lung cancer

99mTc-fanolesomab (CD15) Granulocyte antigen CD15 Appendicitis

111In-antimyosin Antimyosin Acute myocardial infarction, heart

transplant rejection Radiolabeled metabolic substrates

18F-Fluorodeoxyglucose, FDG Tumor viability and metabolism Tumor imaging

Glucose metabolism Brain and cardiac imaging

18F-Fluorothymidine Cell proliferation Tumor imaging and monitoring treatment

11C-choline Cell proliferation Brain tumors

[¹¹C] or¹²³I-methyl tyrosine Protein synthesis, protein upregulation Brain tumors

11C-methionine Amino acid transport Brain and pancreatic tumors

[¹¹C]-thymidine DNA synthesis, cell proliferation Brain tumors [¹⁸F] and¹²³I-fatty acids Myocardial metabolism Cardiac imaging [⁵⁷Co]-vitamin B12 Vitamin B12absorption Pernicious anemia

18F-fluoromisonidazole Hypoxia and oxidative metabolism Tumors selected for radiotherapy

18F-fluoroethyltyrosine(FET) Amino acid transporter Brain tumors

Table 2.3. Radiopharmaceuticals for therapy

Radiopharmaceutical Application Specific tumors

131I-sodium iodide Thyroid function Differentiated thyroid carcinoma

131I-MIBG Adrenergic tissue Colorectal cancer metastatic to liver and

bladder cancer

131I-anti B1 antibody Anti CD22 antigen Lymphoma

90Y-MXDTPA-anti B1 antibody Anti CD22 antigen Lymphoma

32P-chromic phosphate (colloid) Cell proliferation and protein synthesis Peritoneal metastases, recurrent malignant ascites

32P-orthophosphate Cell proliferation and protein synthesis Polycythemia vera

89Sr chloride Exchanges with Ca in bone Palliation of pain due to bony metastases

153Sm-EDTMP Binds to hydroxyapatite Palliation of pain due to bony metastases

117mSn-DTPA Binds to hydroxyapatite Palliation of pain due to bony metastases

186Re-HEDP Binds to hydroxyapatite Palliation of pain due to bony metastases

90Y-DOTA-Tyr³-octreotide Somatostatin receptors Neuroendocrine tumors

90Y-DOTA-lanreotide Somatostatin receptors Neuroendocrine tumors

90Yb-ibritumomab Lymphocyte antigen CD20 Lymphoma

2.4 Radiopharmaceuticals

33

2.5

Mechanism(s) of Radiopharmaceutical Localization

The uptake and retention of radiopharmaceuticals by different tissues and organs involve many different mechanisms, as summarized in Table 2.4. The pharma- cokinetics, biodistribution, and metabolism of the ra- diopharmaceutical are very important to understand- ing the mechanisms of radiopharmaceutical localiza- tion in the organ or tissue of interest. As discussed above under Pathophysiology, the injury to a cell or tis- sue significantly alters the morphology and molecular biology compared with that of normal tissue or organs.

This is especially true of malignant tissue. Compared with normal cells, the tumor cells have very distinct characteristics. These include (a) an increased rate of cell proliferation, (b) altered membrane transport fea- tures associated with blood vessels and tumor cells, (c) altered perfusion within the tumor, (d) altered metabo-

Table 2.4. Mechanisms of radiopharmaceutical locali- zation

Mechanism Radiopharmaceutical

1. Isotope dilution ¹²⁵I-HSA,⁵¹Cr-RBC, and^99mTc-RBC

2. Capillary blockade ^99mTc-MAA

3. Physicochemical adsorption ^99mTc-MDP, HDP

4. Cellular migration ¹¹¹In- and^99mTc-leukocytes,¹¹¹In-platelets 5. Cell sequestration Heat denatured^99mTc-RBC

6. Simple diffusion ¹³³Xe,^81mKr,^99mTc-pertechnegas Diffusion and mitochondrial binding ^99mTc-sestamibi and tetrafosmin Diffusion and intracellular binding ^99mTc-Ceretec and Neurolite Diffusion and increased capillary

permeability

67Ga-citrate 7. Facilitated diffusion and transport,

protein upregulation

[¹⁸F]-FDG, radiolabeled amino acids 8. Active transport Radioiodide,^99mTcO4–,²⁰¹Tl thallous cation

[¹⁸F]-FDG, radiolabeled amino acids Na⁺/K⁺ATPase pump ²⁰¹Tl thallous cation

9. Phagocytosis ^99mTc-colloids in RES and lymph nodes 10. Increased vascular permeability and

capillary leakage

67Ga-citrate, radiolabeled proteins 11. Cell proliferation [¹¹C]-thymidine, [¹²⁴I]-iododeoxyuridine

(IudR),¹⁸F-fluorothymidine (FLT) 12. Metabolic trapping [¹⁸F]-FDG,^99mTc-pertechnetate

13. Metabolic substrates ¹²³I and¹³¹I as sodium iodide,¹²³I-fatty acids 14. Tissue hypoxia and acidic pH [¹⁸F]fluoromisonidazole,⁶⁷Ga-citrate 15. Specific receptor binding

Somatostatin receptors OctreoScan, NeoTect

VIP receptors ¹²³I-VIP

Transferrin receptors ⁶⁷Ga-citrate

Estrogen receptors 16[ -[¹⁸F]fluoro-17q -estradiol (FES) Dopamine D2 receptors [¹²³I]-IBZM,^99mTc-TRODAT

LDL receptors ¹³¹I-6q -iodomethyl-19-norcholesterol (NP-59) Presynaptic adrenergic reuptake [¹³¹I or¹²³I]-MIBG

16. Specific binding to tumor antigens

17. PSMA ProstaScint

CEA CEA-Scan

TAG-72 OncoScint

Cell surface 40-kd glycoprotein Verluma

CD22 Bexaar

CD15 ^99mTc-fanolesomab

Antimyosin ¹¹¹In-antimyosin

lism, (e) altered expression of specific receptors for hormones, and (f) expression of specific tumor-associ- ated antigens.

The mechanisms of radiopharmaceutical localiza- tion may be substrate-nonspecific (not participating in any specific biochemical reaction) or substrate specific (participating in a specific biochemical reaction), de- pending upon the chemistry of the molecule. Many ra- diopharmaceuticals were designed to take advantage of the pathophysiology in order to increase the specificity of the nuclear medicine imaging techniques. Since some radiopharmaceuticals are not specific for a par- ticular disease, the cellular uptake might include a combination of different mechanisms, as in the case of

67

Ga citrate. However, the unique chemistry of each ra-

diopharmaceutical may determine the manner in

which it is transported and retained within a specific

tissue or organ. It is very important to recognize that

since the radiopharmaceutical may have significant

metabolism and degradation in vivo, the observed bio-

distribution and tissue localization may represent the behavior of only radiolabeled metabolic product and not necessarily that of the intact parent radiopharma- ceutical. In addition, the patient’s medication and many other factors may significantly alter the biodistri- bution and tissue localization and retention character- istics of a radiopharmaceutical. The different mecha- nisms of localization are discussed below, using specif- ic examples of the more common radiopharmaceuti- cals.

2.5.1

Isotope Dilution

The dilution principle is based on the concept of “dilut- ing” a radiotracer (or tracer) of known activity (or mass) in an unknown volume. By measuring the degree to which the radiotracer was diluted by the unknown volume, one can determine the total volume (or mass) of the unknown volume. The dilution principle is cur- rently used for a quantitative determination of RBC volume (mass), plasma volume, and total blood vol- ume. It is very important that the radiotracer remain only in the blood volume to be measured. Nondiffus- ible intravascular agents such as

Cr-RBCs are used to measure RBC mass, while

I-HSA is used to measure plasma volume. There is no specific mechanism in- volved other than simple dilution of the radiotracer.

The use of

Tc-RBCs for the measurement of cardiac ejection fraction and gastrointestinal bleeding studies is another application of the dilution principle.

2.5.2

Capillary Blockade

The technique most commonly used to determine the perfusion to an organ depends on trapping the radiola- beled particles (microembolization) in the capillary bed of an organ such as lung, heart, or brain. Pulmo- nary capillaries have a mean diameter of about 8 µm and the precapillary arterioles have a diameter of 20–

25 µm.

Tc-MAA particles generally are in the range of 10 – 50 µm in diameter. Therefore, following intrave- nous injection,

Tc-MAA particles are physically trapped in the arteriocapillary beds of the lung and block the blood flow to the distal regions. Smaller par- ticles pass through the pulmonary capillaries and are extracted by the reticuloendothelial system in the body.

Therefore, the mechanism of localization of particles in lungs is purely a mechanical process, called capillary blockade. The gold standard for the determination of perfusion in experimental animal studies is radiola- beled microspheres, with varying physical half-lives and particle diameters.

2.5.3

Physicochemical Adsorption

Bone scanning with

Tc-labeled phosphonates (MDP, HDP) is extensively used in nuclear medicine to evalu- ate osteomyelitis, arthritis, Paget’s disease, and the bone involvement or metastatic spread to bone in pa- tients with a wide variety of cancers. The

Tc-phos- phonates accumulate in hydroxyapatite (HA) crystal (containing Ca

and phosphate ions) matrix or in the amorphous (noncrystalline) calcium phosphate (ACP).

The principal uptake mechanism of the radiotracer ap- pears to be simply “physicochemical adsorption”.

However, the exact mechanisms involved in the extrac- tion of the radiotracer from the blood through the en- dothelial cells, extracellular fluid, and finally to HA are not known. In contrast to the P-O-P bond in phos- phates, the P-C-P bond in phosphonates is not a sub- strate for alkaline phosphatase and is very stable in vi- vo. Primary bone tumors such as osteogenic sarcomas avidly accumulate bone agents because of the produc- tion of bone matrix in extraosseous tissue. Metastatic deposits that produce a vigorous osteoblastic response will appear as hot spots in a bone scan, while the lesions that generate osteolytic reactions may not accumulate the bone agent [6]. The bone scanning agents may also be taken up occasionally in soft tissues. The primary underlying factor responsible for the uptake of these tracers is excess calcium in soft tissue. Cell hypoxia and cell death would lead to increased deposition of calci- um phosphates in the extracellular fluid. The uptake of

99m

Tc-phosphonates in the soft tissues is believed to be due to chemisorption on the surface of calcium salts.

The localization of bone-seeking radiotracers in in- creased amounts at the tumor-bone interface provides the basis for the use of radionuclides in the treatment of bone pain. Several radiopharmaceuticals (Table 2.3) are indicated for relief of pain (bone pain palliation) in patients with confirmed osteoblastic bone lesions. The exact mechanism of action of relieving the pain of bone metastases is not known, however.

2.5.4

Cellular Migration and Sequestration

111

In-oxine- or

Tc-HMPAO-labeled autologous mixed leukocytes (predominantly neutrophilic poly- morphonuclear leukocytes, PMNs) are routinely used to image various inflammatory diseases and infectious processes. The inflammatory reaction is a well-de- scribed sequence of events in response to an infection.

In an acute infection, within the first 6 – 12 h the pre- dominant cells infiltrating a site of infection are the PMNs. Following intravenous administration of radio- labeled leukocytes, the labeled cells migrate to the site of infection, similar to the circulating leukocytes. The

2.5 Mechanism(s) of Radiopharmaceutical Localization

35

Membrane

99mTc I,I-ECD(COOH2CH3)2 99mTc I,I-ECD(COOH2CH3)2

99mTc I,I-ECD(COOH)₂+2CH₃CH₂OH esterase

Brain Blood

leukocytes migrate to the site of infection/inflamma- tion because they are attracted by the immediately gen- erated chemotactic factors, such as complement sub- components. In a similar manner,

In-platelet locali- zation at the site of active thrombus formation also in- volves simple cellular migration, since platelets play a major role in thrombus formation.

Accessory splenic tissue can develop after splenec- tomy. Heat-damaged

Tc-RBCs are more specific for the detection of accessory splenic tissue. Following in- travenous administration, the spleen sequesters the heat-damaged RBCs in the same way that old and dam- aged circulating RBCs are normally removed.

2.5.5

Membrane Transport

The cell membrane consists of a lipid bilayer that is not miscible with either the extracellular fluid or the intra- cellular fluid and provides a barrier for the transport of water molecules and water-soluble substances across the cell membrane. The transport proteins with in the lipid bilayer, however, provide different mechanisms for the transport of molecules across the membrane.

Membranes of most cells contain pores or specific channels that permit the rapid movement of solute molecules across the plasma membrane. Channels are selective for specific inorganic ions, whereas pores are not selective. Plasma membranes contain transport systems (transporters) that involve intrinsic membrane proteins and actually translocate the molecule or ion across the membrane by binding and physically mov- ing the substance. Transporters play important roles in the uptake of nutrients, maintenance of ion concentra- tions, and control of metabolism. Transport through the lipid bilayer or through the transport proteins may involve simple diffusion, passive transport (facilitated diffusion), or active transport mechanisms. Certain macromolecules may also be transported by vesicle for- mation, involving either endocytosis or exocytosis mechanisms.

Fig. 2.1. With the labeling of red blood cells (RBCs) with^99mTc whether in vitro or in vivo, the

99mTcO4freely diffuses in and out of the RBCs, but in the presence of stannous ion it is reduced intracel- lularly, where it reacts with hemo- globin (HB) to form^99mTc-Hb. The

99mTc-Hb does not diffuse out of the RBCs as shown in Fig. 2.2 2.5.5.1

Simple Diffusion

The mechanism of localization of many radiopharma- ceuticals in target organs involves a simple diffusion process. The direction of movement of the radiotracers by diffusion is always from a higher to a lower concen- tration, and the initial rate of diffusion is directly pro- portional to the concentration of the radiotracer. A net movement of molecules from one side to another will continue until the concentration on each side is at chemical equilibrium.

The gases used for ventilation studies, such as

Xe,

127

Xe, and

Kr, are inert lipophilic gases. Following their administration through inhalation, these gases are distributed within the lung air spaces by diffusion, proportional to ventilation. The distribution, however, is interrupted in obstructive airways. The gases pass from the lungs into the pulmonary venous circulation and are released through the lungs by the mechanism of alveolar capillary diffusion. Similarly, distribution of

99m

Tc-Technegas within the lung also involves diffu- sion. By contrast, the irregular distribution of

Tc- DTPA aerosol preparation within the lung is due mostly to gravity sedimentation depending on particle size.

2.5.5.1.1

Diffusion and Intracellular Metabolism/Binding

The blood-brain barrier (BBB) plays an important role

in the mechanism of localization of many radiophar-

maceuticals in the brain. The endothelial cells of the ce-

rebral vessels form a continuous layer without gap

junctions, preventing diffusion of water-soluble mole-

cules. In certain pathological conditions, the BBB is

disrupted, allowing water-soluble molecules to diffuse

from the blood into brain tissue. Traditionally, the

brain scan was performed with such radiopharmaceu-

ticals as

Tc-pertechnetate and

Tc-DTPA, that dif-

fuse freely in the extracellular fluid and can accumulate

in lesions with defects in BBB. The intact BBB does al-

low the transport of small molecules across the plasma

Red cell membrane

+Hgb Intracellular Extracellular

99mTcO4

- 99m

TcO4 -+Sn²⁺

99mTcHgb

Fig. 2.2. Intracellular binding of^99mTcO4to Hgb

membrane of the neuron by facilitated diffusion. Some small, neutral molecules, however, can cross the BBB depending on their relative lipid solubility. Brain perfu- sion-imaging agents such as

I-IMP (N-isopropyl-p- iodoamphetamine),

Tc-HMPAO (Ceretec), and

99m

Tc-ECD (Neurolite) are lipophilic radiotracers that cross the BBB via passive diffusion. The extraction of these tracers by the brain tissue is proportional to re- gional cerebral blood flow (rCBF). The retention of these tracers within the neuronal tissue following diffu- sion and extraction is assumed to be due to intracellu- lar binding or metabolic degradation to polar metabo- lites or charged complexes that cannot be washed out of the cell by back-diffusion as exemplified by the cellular retention of

Tc-ECD (Fig. 2.1) and labeling of

Tc- pertechnetate with RBC (Fig. 2.2). With

TC-ECD the radiotracer freely diffuses into the brain tissue, where it is hydrolyzed by the action of esterase to an acid which is trapped in the brain tissue. For

TcO4, the tracer is reduced intracellulary by the circulating stannous ion and the reduced

Tc binds to Hgb to form

TcHgb that does not diffuse out of the brain tissue.

2.5.5.1.2

Diffusion and Mitochondrial Binding

A number of lipophilic, cationic

Tc radiopharma- ceuticals (sestamibi, tetrofosmin, and furifosmin) have been developed for imaging myocardial perfusion. Al- though

Tc-sestamibi is cationic, similar to

Tl

, the transport of this agent through the cell membrane in- volves only passive diffusion since the transport pro- cess is temperature dependent and nonsaturable [7].

The myocardial cell uptake of

Tc-sestamibi was ini- tially considered to be due to binding of the cell mem- brane to lipid components. It was later shown that in- tracellular binding of

Tc-sestamibi was associated mainly with mitochondria. Using cultured chicken my- ocytes, Piwnica-Worms et al. [8] observed that the cel- lular entry of

Tc-sestamibi is related to the mito- chondrial metabolism and the negative inner mem-

brane potential of the mitochondria. The mitochondri- al retention of

Tc-sestamibi, however, is not organ or tumor specific, but appears to be a mechanism com- mon to most types of tissue. The intracellular levels of Ca

in normal cells are significantly low. However, with irreversible ischemia, extracellular calcium enters the cell and is sequestered in the mitochondria, result- ing in mitochondrial destruction. The increased calci- um concentration in mitochondria blocks

Tc-sesta- mibi binding to mitochondria.

A number of investigators have reported the diag- nostic potential of

Tc lipophilic cationic complexes for imaging parathyroid adenomas, osteosarcomas, and tumors of the brain, breast, lung, and thyroid.

The mechanisms of uptake of sestamibi and tetro- fosmin have been compared to that of

Tl in tumor cell lines [9]. While the

Tc agents are associated with mitochondria,

Tl remains in the cytoplasmic com- partment. There were significant differences between sestamibi and tetrofosmin regarding intracellular lo- calization based on in vitro studies. While 90% of total sestamibi was associated with mitochondria, most of the tetrofosmin accumulated in the cytosolic fraction [9]. The uptake and retention of lipophilic

Tc cation- ic agents by tumor cells appear to be related to the back-diffusion or efflux of the tracer from the cell.

There is now extensive evidence suggesting that the transport of the tracer out of the tumor cell is mediated by P-glycoprotein (Pgp), a 17-kd plasma membrane li- poprotein encoded by the human multidrug resistance (MDR) gene. Piwnica-Worms et al. [10, 11] demon- strated that sestamibi is a transport substrate for Pgp and is useful for imaging Pgp expression.

2.5.5.1.3

Diffusion and Increased Capillary and Plasma Membrane Permeability

67

Ga-citrate has been shown to localize in a variety of tumors and inflammatory lesions. In the past three de- cades, a number of investigators have reported various physical and biochemical factors responsible for the tu- mor uptake of

Ga-citrate. However, there is still no general agreement on the exact mechanisms of locali- zation in tumors, as indicated by many review articles [12, 13]. Following intravenous administration of car- rier-free

Ga as gallium citrate,

Ga is bound exclu- sively to the two specific metal-binding sites of iron- transport glycoprotein, transferrin in normal plasma, and is transported to normal tissues and tumor sites predominantly as

Ga-transferrin complex [14]. At a physiological pH of 7.4, gallium may also exist as a sol- uble gallate ion, Ga(OH)

. The volume of distribution of

Ga in patients is about 23 l, suggesting that gallium is extensively distributed in the extravascular space [13]. While the

Ga-transferrin complex is slowly

2.5 Mechanism(s) of Radiopharmaceutical Localization

37

transported through the capillary vessel wall, the non- transferrin-bound, free gallium rapidly leaves the blood compartment and equilibrates with the intersti- tial fluid of normal and tumor tissue. The increased capillary permeability and the expanded extracellular space of tumor tissue would also augment the transport of macromolecules such as transferrin (80 kd) across the leakier tumor blood vessels. Increased transferrin concentration within the interstitial fluid of the tumors would also lend support to the role of increased capil- lary permeability in

Ga tumor localization. The mechanisms involved in the uptake of

Ga by tumor cells are very complex, since a variety of factors appear to affect transport and retention of

Ga within the tu- mor tissue. Based on in vivo studies, Hayes et al. [15]

concluded that the initial entry of

Ga into tumor tis- sue involves simple diffusion of the unbound or loosely bound form of

Ga, whereas its uptake by normal soft tissues is strongly promoted by its binding to transfer- rin. The increased permeability of the tumor cell mem- brane compared with normal cells may also account for increased diffusion of non-transferrin-bound gallium species into cells. The accumulation of

Ga within tu- mor cells is very much dependent upon the intracellu- lar binding of

Ga to iron-binding proteins such as lac- toferrin and ferritin or other higher-molecular-weight molecules which can chelate gallium with greater affin- ity, thereby preventing back-diffusion of free gallium species [13].

2.5.5.2

Facilitated Diffusion

18

F-fluorodeoxyglucose (FDG). All cells use glucose to generate metabolic energy. For brain tissue, glucose is the primary source of energy, but in the heart glucose becomes the primary source of energy for ischemic myocardium. Tumor cells have increased rates of an- aerobic and aerobic glycolysis compared with most normal tissues. Glucose is transported into the cell across the plasma membrane by facilitated diffusion, mediated by members of the glucose transporter (Glut) protein family (Glut1 – 6) [16]. Similar to glucose, FDG is also transported into normal and malignant cells by facilitated diffusion.

Hepatobiliary Agents. Evaluation of hepatocyte function using radiopharmaceuticals that are excreted via biliary secretion is another example of a carrier- mediated transport mechanism. Lipophilic organic an- ions with nonpolar groups (favoring plasma protein binding) having molecular weights in the range of 600 – 1000 are predominantly removed from the body via bil- iary excretion. Following intravenous administration,

99m

Tc-disofenin (Hepatolite) and

Tc-mebrofenin (Choletec) diffuse through pores in the endothelial lin-

ing of the sinusoids and bind to the anionic membrane- bound carriers on the hepatocyte. The hepatic uptake is facilitated by carrier-mediated, non-sodium-depen- dent, organic anionic pathways similar to that of biliru- bin. Subsequent biliary excretion of the radiotracer is relatively passive and involves following the flow of bile through the biliary tree. The bile may be stored and concentrated temporarily in the gallbladder or excreted directly into the intestine. Since bilirubin is excreted by the same hepatocyte transport system, higher serum bilirubin levels may have a significant effect on the bio- distribution and hepatic excretion of radiopharmaceu- ticals. Of the

Tc-iminodiacetate (IDA) derivatives,

99m

Tc-mebrofenin combines the best characteristics of high hepatic uptake, low urinary excretion, fast blood clearance and hepatocellular transit, and has the high- est degree of resistance to the competitive effects of bil- irubin as measured in isolated hepatocytes.

2.5.5.3

Active Transport

Active transport involves translocating a solute mole- cule through a cell membrane against its concentration gradient and requires the expenditure of some form of energy. Active transport is driven by either hydrolysis of ATP to ADP (primary active transporters) or utiliza- tion of an electrochemical gradient of Na

or H

(sec- ondary active transporters) across the membrane. If the energy source is inhibited or removed, the trans- port system will not function.

Radioiodide and

Tc-Pertechnetate Anions. Thy- roid tissue selectively traps certain anions, such as I

, TcO

, and ClO

, by an active transport mechanism us- ing the same pathway; hence they are the competitive inhibitors of each other. The clinical implication is that iodinated contrast agents or iodine containing medica- tions may interfere with the accumulation of

TcO

in the thyroid, thereby leading to poor image quality.

However, only iodide is used by the thyroid gland to

synthesize thyroid hormones, while the other anions

diffuse out of the gland. In addition to thyroid tissue,

the salivary glands, stomach, bowel, and genitourinary

tract show significant uptake (secretion) of radioiodide

and pertechnetate. Radioiodide is rapidly absorbed

from the GI tract after oral administration and is accu-

mulated in thyroid tissue over the next 24 h. This up-

take may be affected by TSH levels, thyroid and nonthy-

roid medications, and the total body iodine pool. Nor-

mal thyroid tissue has a very high affinity for iodide,

while thyroid cancer and metastases accumulate iodide

less readily. Papillary and follicular cancers arise from

the thyroid follicular cells and retain to a certain extent

the ability to trap iodide. By contrast, medullary carci-

noma of the thyroid arises from the parafollicular, or C,

cells of the thyroid and does not accumulate radio- iodide.

201

Thallous Chloride. Since the thallous ion (Tl(OH)

) acts as an analogue of the K

ion,

Tl is used to image myocardial perfusion in order to evaluate the extent of myocardial ischemia and/or infarction. Positron emit- ter

Rb – a monocation, like potassium – is also useful for imaging myocardial perfusion. The myocardial up- take of thallium and rubidium involves active cation transport mechanisms including both passive diffu- sion and ATP or energy-dependent pathways [19].

The diagnostic value of

Tl for imaging brain tu- mors, osteosarcomas, low-grade lymphomas, Kaposi sarcomas, and parathyroid tumors is well established.

Accumulation of

Tl in the tumor is a function of tu- mor blood flow and increased cell-membrane perme- ability. The tumor cell uptake of

Tl also appears to be due to an active transport system involving the Na

/ K

ATPase pump within cell membranes. Based on studies with murine Ehrlich ascites tumor cells, Ses- sler et al. [20] have demonstrated that the cellular up- take of

Tl is inhibited by ouabain, digitalis, and furo- semide, which block the Na

/K

pump. The drug oua- bain blocks only the ATPase-dependent Na

/K

pump while furosemide can also block a chloride co-trans- port system. Sessler et al. [21] observed that the inhibi- tion of

Tl uptake by ouabain and furosemide was ad- ditive and suggested that

Tl uptake into the cell may involve two separate transport mechanisms. In addi- tion, even after the blockade of Na

/K

ATPase and chloride transport systems, a minimal amount of

Tl was taken up by the cells and this transport mecha- nism may be mediated by the calcium-dependent ion channel [21].

Renal Agents. Each kidney is made up of about 1 mil- lion nephrons, each with two components, a glomeru- lus and a long tubule that has three segments: the proxi- mal tubule, loop of Henle, and distal tubule. One of the major functions of the kidney is excretion of waste ma- terials by glomerular filtration and tubular secretion.

Depending on the needs of the body, some of the mole- cules are subsequently reabsorbed back into the blood.

Tubular reabsorption is the movement of fluids and solutes (Na

and glucose) from the tubular lumen to the peritubular capillary plasma, while tubular secretion involves transport of molecules (ammonia and hydro- gen ions) from the plasma of peritubular capillary to the tubular lumen. Glomerular filtration (GFR) pro- vides the best estimate of functioning renal tissue. The measurement of GFR requires a molecule such as inulin that has a stable plasma concentration and is freely fil- tered in the glomerulus and not secreted or reabsorbed by the tubule. The radiotracers most commonly used for measurement include

I-iothalamate,

Tc-DTPA

or

Cr-EDTA, since they meet the necessary require- ments for glomerular filtration. No specific transport mechanisms are involved in the filtration process, and the GFR is determined by the sum of hydrostatic and colloid osmotic forces across the glomerular mem- brane. Radiopharmaceuticals such as radioiodinated hippuran and

Tc-mercaptoacetyltriglycine (MAG

) are partly filtered in the glomerulus but mostly excret- ed by tubular secretion. Compared with radioiodinated hippuran (30% by glomerular filtration), most of

99m

Tc-MAG3 is bound to plasma proteins and only about 10% may undergo glomerular filtration. These carboxylate substrates are actively transported by the renal hippurate anionic transport system of the proxi- mal convoluted tubule cells [23].

2.5.5.4 Phagocytosis

Most of the

Tc-sulfur colloid (SC) particles are in the range of 0.1 – 1.0 µm. Following intravenous adminis- tration, particles are able to leave the circulation via the sinusoidal type capillary structures in the liver, spleen, and bone marrow. Specific serum proteins known as opsonins may interact and provide a proper coating to the particles so that they may be recognized by recep- tors on the phagocytic cell surface. The cells of the re- ticuloendothelial system (RES) engulf the colloid parti- cles and remove them from circulation. Kupffer’s cells (macrophages in liver sinusoids) and reticular cells (macrophages in spleen) accumulate the particles by phagocytosis. Cold lesions identified on a liver scan with

Tc-SC may be due to an intrahepatic tumor dis- placing the usual distribution of RES cells. Similarly, radiation damage in liver and bone marrow is seen as cold areas due to decreased RES function.

Recently,

Tc-SC has been used extensively in lym- phoscintigraphy in order to identify a “sentinal node”

(first lymph node to receive lymphatic drainage from a tumor site) in patients with breast cancer and melano- ma [24]. If radiocolloid is introduced into the intersti- tial fluid, it drains into the lymphatic vessels and then into regional lymph nodes. Colloid particles smaller than 0.1 µm show rapid clearance from the interstitial space into lymphatic vessels and significant retention in lymph nodes. While normal lymph nodes appear as hot spots, cancerous nodes do not sequester colloids, resulting in false-negative identification. Because of their small particle size,

Tc-antimony sulfide-col- loid (0.002 – 0.015 µm) and

Tc-human serum albu- min, or nanocolloid (0.01 – 0.02 µm) are ideal for lym- phoscintigraphy studies. Since these agents are not available in the United States, filtered (using a 0.2-µm filter)

Tc-SC preparation is being used for sentinal node detection [24].

2.5 Mechanism(s) of Radiopharmaceutical Localization

39

Membrane

hexokinase Tissue Blood

18FDG ¹⁸FDG

18FDG-6-PO4

2.5.5.5

Receptor-Mediated Endocytosis

A common pathway for tumor cell uptake of

Ga and

59

Fe via a transferrin receptor was initially proposed by several investigators [25], who suggested that

Ga lo- calization in tumors involves endocytosis of the

Ga- transferrin-receptor complex. In vitro studies clearly demonstrated that transferrin, at low concentrations (

0.1 mg/ml), stimulated and increased

Ga uptake by tumor cells. However, in vivo studies with animal tu- mor models provided conflicting results regarding the role of transferrin receptors in

Ga localization [12]. In addition,

Ga tumor uptake was also observed in tu- mor-bearing mice with congenitally absent transferrin (hypotransferrinemic) [26]. Although there is some ev- idence that the number of transferrin receptors in tu- mor cells may be increased tenfold compared with nor- mal cells [13], the exact connection between transfer- rin receptors and

Ga tumor uptake in vivo has not been established.

2.5.6.

Metabolic Substrates and Precursors

Cancer cells have an altered metabolism compared with normal cells. As a result, cancer cells use more glu- cose than normal cells. Due to the increased rate of cell proliferation, the protein and DNA synthesis is aug- mented and the cancer cells need to transport in- creased amounts of precursors such as amino acids and nucleotides. A number of radiopharmaceuticals were developed based on the increased demand of metabolic substrates of tumor cells.

Metabolic Trapping of FDG.

F-2-deoxy-2-fluoro-d- glucose (FDG) was developed in 1977 to measure local cerebral glucose utilization using PET [27]. In 1980, Som et al. [28] demonstrated that FDG accumulated in a variety of transplanted and spontaneous tumors in animals. Similar to d-glucose, FDG is transported into the cell by facilitated diffusion and is phosphorylated by hexokinase to FDG-6-phosphate (Fig. 2.3).

In the next step of glycolysis, the enzyme glucose-6- phosphate isomerase does not react with FDG-6-phos- phate due to very strict structural and geometric de- mands. As a result, the very polar FDG-6-phosphate is trapped in the cytoplasm [29]. FDG-6-phosphate may be converted back to FDG, but the enzyme glucose 6- phosphatase, which is responsible for this reaction, is either at very low levels or absent in cancer tissue. FDG- PET is now being extensively used for an increasing number of clinical indications at different stages of can- cer, e.g., diagnosis, staging, monitoring of response to therapy, and finally detection of recurrence. In addi- tion, FDG accumulates in granulomatous tissue and

Fig. 2.3. Intracellular phosphorylation of ¹⁸FDG to¹⁸FDG-6- PO4by hexokinase

macrophages infiltrating the areas surrounding necrot- ic tumor tissue [30] and may also have a role to play in imaging areas of infection/inflammation.

2.5.6.1

Precursors: Radiolabeled Amino Acids

Since amino acids are the biological building blocks of proteins, radiolabeled amino acid uptake within tu- mors may reflect the increased protein synthesis rate of proliferating tumor cells or simply an increased rate of amino acid transport across the tumor cell membrane [31]. Despite its complex biochemistry and in vivo me- tabolism, methionine has been the most widely used amino acid tracer, in the form of l-[methyl-

C]methio- nine. The predominant mechanism of methionine tu- mor uptake reflects the increased rate of active mem- brane transport process rather than the rate of protein synthesis [32]. Since tyrosine reflects the protein syn- thesis rate, radiolabeled tyrosine and a number of tyro- sine analogs have been evaluated [33]. These include l-[1-

C]tyrosine, l-[2-

F]fluorotyrosine, l-4-[

F]fluo- ro-m-tyrosine, and l-[3-

F]-a-methyltyrosine (FMT).

Among these tracers, FMT is relatively easy to synthe- size and displays high in vivo stability, with 75% of the injected dose in the unmetabolized form in the circula- tion [33].

Recently, the tyrosine analog L-O-[2-

F]fluorethyl-

tyrosine (FET), which is not incorporated into proteins

but nevertheless transported by an active transport

mechanism, was developed [34]. FET is stable in vivo

with fast brain and tumor uptake kinetics, and the bio-

distribution reflects that of an unnatural amino acid

[34]. Radiolabeled natural amino acids and analogs al-

so exhibit high uptake in normal brain tissue. A num-

ber of unnatural nonmetabolized amino acids can be

used as substrates for active transport with minimal

accumulation by normal brain tissue. Labeled with

positron emitters, these compounds have been investi-

gated as tumor-imaging agents. Among these tracers,

[

C]a-aminocyclobutane carboxylic acid (ACBC) and [

F]FACBC analogs showed intense uptake in such brain tumors as astrocytomas and glioblastomas [33].

2.5.7

Tissue Hypoxia

Imaging. Hypoxia may result from either insufficient regional perfusion (acute or transient hypoxia), as in myocardium, or insufficient oxygen diffusion (chronic hypoxia), as in tumors. Since hypoxia cannot be pre- dicted, noninvasive techniques for identifying hypoxic regions in tumor, myocardium, and brain tissue are be- ing developed. The compound 2-nitro in misonidazole (MISO) is transported into the cell by diffusion. In the cytoplasm, the nitro group (NO

) undergoes one elec- tron enzymatic reduction to the free radical anion [35].

In normoxic cells, this reaction step is reversed by in- tracellular oxygen and the oxidized molecule diffuses out of the cell. In hypoxic tissue, the free radical is fur- ther reduced to a reactive species, hydroxylamine, and then to an amine [35]. Free radicals are attached irre- versibly to cellular macromolecules and are retained within the cell. Reduction of these molecules occurs in all tissue with viable enzymatic processes, but reten- tion occurs only in those tissues with low oxygen ten- sion.

A number of radiolabeled compounds incorporat- ing a 2-nitroimidazole moiety to image tumor hypoxia have been developed.

F-fluoromisonidazole (FMISO) is probably the most extensively studied hypoxia-selec- tive radiopharmaceutical [36]. In order to develop PET tracers for hypoxia imaging, radiolabeled agents of copper have been investigated, since copper has an amenable coordination and electrochemistry that would lend itself to redox-mediated trapping in cells.

One of these compounds,

Cu-ATSM (Cu-diacetyl-

-methylthiosemicarbazone) has been shown to be selectively trapped in hypoxic tissue but rapidly washed out of normoxic cells [37].

Among the iodinated compounds, successful imag- ing of tumor hypoxia has been reported using a sugar containing the MISO derivative

I-iodoazomycin ara- binoside (IAZA) [38]. Significant in vivo deiodination, however, limits the clinical usefulness of this com- pound. A

Tc-labeled hypoxic imaging agent, a pro- pylene amine oxime (PnAO) derivative of 2-nitroimid- azole also known as BMS181321, showed hypoxia selec- tivity in tumor models but has slow clearance due to high lipophilicity [39]. It was recently reported that a complex of core ligands without the nitroimidazole group labeled with

Tc also showed very high tumor- hypoxia selectivity. A prototype formulation of one of these compounds,

Tc-HL91 (4,9-diaza-3,3,10,10-te- tramethyldodecan-2,11-dione dioxime), has demon- strated uptake in a variety of tumors [40].

Hypoxia and Tumor pH. Increased glucose metabo- lism of tumor cells was initially recognized in 1925 by Warburg [41], who observed that tumor cells have in- creased rates of anaerobic and aerobic glycolysis com- pared with most normal tissues. In glucose metabo- lism, the initial reaction sequence, known as glycolysis, takes place in the cytoplasm where glucose is converted to two molecules of pyruvate. Under anaerobic condi- tions (hypoxia), this mechanism is unavailable; pyru- vate is converted to lactic acid by lactate dehydrogenase (LDH) and accumulates. Consequently, the pH of the tumor tissue is lightly acidic [41], compared with nor- mal tissue pH of 7.4. The acidic pH of the tumor tissue may possibly play a significant role in the mechanism of

Ga localization in tumors. The stability of the

Ga- transferrin complex is very much dependent upon bi- carbonate concentration and pH; decreasing either bi- carbonate or pH would help more

Ga to dissociate from transferrin [42] and would help to generate more free gallium species. The pH of the interstitial fluid of tumors is slightly acidic compared with the normal tis- sue, and reducing tumor pH by enhancing anaerobic glycolysis in tumor-bearing rats actually increased

Ga uptake by tumors [42].

2.5.8

Cell Proliferation

In normal tissue there is a balance between cell growth and cell death. Within a tumor, growth is favored. Until recently, increased mitotic rate, cell proliferation, and lack of differentiation were regarded as the main fac- tors responsible for accelerated growth of malignant tissue. Most benign tumors grow slowly over a period of years, but most malignant tumors grow rapidly, sometimes at an erratic pace. In general, the growth rate of tumors correlates with their level of differentia- tion; thus most malignant tumors grow more rapidly than benign tumors do. Therefore, there is increased mitotic activity in tumor tissue. The number of cells in the S-phase of cell cycle is also higher compared with normal cells. As a result, there is an increased require- ment of substrates (nucleotides) for DNA synthesis.

Nucleotide incorporation into DNA in tumor tissue de- termined in vitro using [

H]-thymidine (thymidine la- beling index) is a measure of tumor proliferation [43].

11

C-Thymidine has been used for many years as a PET tracer to image tumors of the head and neck [44]. Due to the rapid metabolism of this tracer in blood, howev- er, the tumor uptake of

C-thymidine is not optimal for imaging studies and quantitation is difficult.

I-5-io- do-2’-deoxyuridine (IudR), an analog of thymidine (TdR), has recently been developed by replacing the 5- methyl group with an iodine atom [45]. Within the tu- mor cell, IudR is phosphorylated and incorporated in DNA. The therapeutic potential of IudR labeled with

2.5 Mechanism(s) of Radiopharmaceutical Localization

41