A review on evaluation of technetium-99m labeled radiopharmaceuticals

Abstract

In the past few years, substantial progress has been made in technetium chemistry, including the synthesis of a variety of ^99mTc-radiopharmaceuticals. This synthesis can be made feasible by using suitable reducing agents, highly specific ligands, appropriate buffer, and specific pH etc., which results in high radiochemical purity, minimum labeling time, commercial expediency of ⁹⁹Mo/^99mTc generator and high biological efficacy. ^99mTc-radiopharmaceuticals have confirmed their worth in every span of life, especially in clinical and medical applications. Now ^99mTc based pharmaceuticals are being used as diagnostic agents for a large number of infections caused by bacteria or any pathogens, tumors, cancers, ulcers etc. In this review, we discuss synthesis of a variety of ^99mTc carrying biological molecules (antibiotics/antibodies/peptides/amino acids/macro and micro-organic molecules) along with their applications, to overview key innovations.

Introduction

In radiopharmaceuticals, the radioactive tracers are the main components to examining the function of body systems. Many radiopharmaceuticals are available for imaging purposes, which differ in terms of their physical characteristics, bio-distribution and radiation exposure. Medical images provide very helpful information to medical specialists for taking the important and right decision for diagnosis and therapeutic action. The overwhelming applications of radioisotopes in every span of life like agriculture, industry, chemistry, biology and nuclear medicine have made them critically significant. In some diseases radiopharmaceuticals can identify medical abnormalities at an early stage than other diagnostic tests. Synthesis of new radiolabeled agents (radiopharmaceuticals) is the major field of interest of nuclear medicine. Many radionuclides exist in nature, but naturally occurring isotopes do not have suitable half-life for medical use. Radiopharmaceuticals are being used in medical applications for molecular imaging and treatment of various infections, cancer and tumor [1–5]. As far as molecular imaging is concerned, it is a distinct technique capable to visualize, characterize and measure the biological processes at the molecular and cellular level, in humans and other living systems [6–13]. Nonetheless, the major radionuclide used for preparing diagnostic radiopharmaceuticals today is ^99mTc, owing to its physical and chemical properties [14–16].

Salient features of ^99mTc

Depending on clinical requirements, gamma or positron emissions, radiolabeling approaches, kinetics and coordinating systems, a variety of radioisotopes have been explored to develop radiopharmaceuticals. Among these, ^99mTc based radiopharmaceuticals have confirmed their significance with ideal physical characteristics (t_1/2 6 h, photon energy 140 keV, no corpuscular radiation), high radiochemical purity, minimum labeling time (10–30 min at room temperature sometimes), low cost, commercial expediency from ⁹⁹Mo/^99mTc generator and high biological efficacy (maximum assimilation by target organ and favorable pharmacokinetics). So more than 80 % of radiopharmaceuticals being used for diagnostic purposes contain ^99mTc [5, 17]. Technetium (^99mTc) has ideal energy of photons which is able to go inside the tissue and it can be detected easily. Due to the short half-life, it reduces the internal radiation hazard and has high limit of intake as compared to other radioisotopes commonly used in laboratories. The chemistry of ^99mTc is very similar to Re because it is located in the periodic table near Rhenium element. Technetium is the 43 element in the periodic table and it is the member of transition metals group VIIB. The electron configuration of technetium is 4d⁵ 5s². Technetium has seven electrons in its outer most shell just like Krypton’s noble gas configuration and enthusiastically loses these electrons to yield the plus seven oxidation state of pertechnetate (${\text{TcO}}_{4}^{ - }$). It has distinguished coordination chemistry with a range of oxidation states between +1 and +7. It facilitates synthesis of technetium based radiopharmaceuticals with diverse ligand environments (O–, C–, Se, N–, P–, S– donor centers and their combinations) [18–31]. Sometimes this diversity does not assist reliable control of the oxidation state and stability of the complexes. On the other hand, it is a matter of fact that this diversity facilitates extensive opportunities for modifying technetium complexes, their structure and properties i.e. altering total charge of the complex, lipophilicity etc. [12, 13, 22–27]. Radiopharmaceuticals are frequently being synthesized with compounds of ^99mTc in oxidation states +1, +3, +4, and +5, using ⁹⁹Mo/^99mTc generator, and suitable reducing agents like SnCl₂·2H₂O, SnF, HCl, NaBH₄, Na₂S₂O₄, Zn dust and FeSO₄ etc. From a generator, ^99mTc is eluted in the form of Na^99mTcO₄. Here negatively charged pertechnetate ion (${}^{{99{\text{m}}}}{\text{TcO}}_{4}^{ - }$) comprises ^99mTc with +7 oxidation state. But in this form, ^99mTc cannot make a stable complex with ligands, peptides or related molecules. Thus, it is necessary to lower the hepta valency of ^99mTc [32–39]. This lowering of oxidation state is accomplished by using suitable reducing agent, specific ligand(s), and most probably the reaction conditions [40–45]. Previous studies revealed efficient labeling of ${}^{{99{\text{m}}}}{\text{TcO}}_{4}^{ - }$ and [^99mTc (H₂O)₃ (CO₃)]⁺ with a variety of bidentate and tridentate biologically active ligands having amine, N-heterocycles, aromatic and/or carboxylic donors [6–10, 21, 46]. Due to the ease of access and labeling, high specificity, rapid assimilation at infection or tumor site, i.e. early diagnosis, rapid blood clearance, high target to non-target ratio, less antigenicity, low toxicity, and high compatible half-life of ^99mTc, it is a more desirable labeling radioisotope as compared to others for diagnostic purposes [32]. Also ^99mTc complexes with antibiotics, drugs, peptides, nucleobases like purine and pyrimidine, amino acids etc. have proven their worth in many biochemical systems. These complexes either intercalate DNA or interfere with DNA replication machinery to intervene cancer, tumor, infection etc. The potential to incorporate this radionuclide (^99mTc) into different targeting determinants has been the prime concern in developing specific diagnostic radiopharmaceuticals [5, 16]. Usually radiopharmaceuticals comprising ligands containing N- and S-centers prove to be the best for diagnosis of renal function [6], while that with O-centers are best for myocardial imaging [7]. Those with S- or P-centers are good for CNS receptor imaging, heart imaging and bone scintigraphy [8, 9, 47].

Factors affecting percentage labeling yield

In developing radiopharmaceuticals, maximum labeling yield and radiochemical purity are the main concern of a researcher. Several parameters including pH level, concentration of reducing agent (SnCl₂) and coordination moiety i.e. ligand in the solution, and boiling time are optimized for good labeling yield and stable complex. Stability of a labeled moiety is of worth importance in terms of shelf-life and biodistribution. After labeling, in vitro stability, while in vivo stability after injecting in a living body, are of major concern [48–54]. As the radio isotopic atoms of the ligand moieties dissociate, the concentration of the labeled compounds in the shelf decreases. Sometimes such dissociation may continue or even increase in the in vivo domain. As it is much feasible for dissociated radioactive atoms to accumulate in different organs/tissues, the compounds with low labeling yields and/or substantial instability inside the body would not give desired biodistribution. Stability of radiolabeled compound is accomplished by temperature, pH and light etc. [10–13, 22]. An optimum pH with appropriate buffer solution plays significant role to acquire maximum radiochemical yield. Usually Phosphate buffer with pH 7 (6–8 in some cases) is found to be the best for a large number of systems [23–27, 55–59]. It is recommended that the injectable radiopharmaceutical should have pH compatible to blood pH (7.4) [12]. Also the selection of suitable reducing agent and its appropriate concentration is the basic requirement to get maximum radiochemical yield. Technetium in the form of pertechnetate ion (${}^{{99{\text{m}}}}{\text{TcO}}_{4}^{ - }$) with +7 oxidation state is actually nonreactive in nature and must be reduced to accelerate labeling reactions. Hydrated stannous chloride (SnCl₂·2H₂O) is found to be effective in synthesizing most of the radiopharmaceuticals. It is observed that an increase in concentration of SnCl₂·2H₂O facilitates increased formation of colloids that leads to decreased yield of the labeled complex [13]. As far as concentration of the starting material (ligands) is concerned, an increase in concentration results in maximum incorporation of ^99mTc because of minimum limit to the volume used [22–25].

^99mTc-labeled antibiotics

Pathogens (bacteria, viruses, parasites, fungi etc.) are regarded as the main source of variety of severe infectious diseases that may lead to mortality or morbidity. Primordial detection and recognition of the infection site allows prompt and successful treatment. Mostly delayed diagnosis of internal infections halts effective treatment and sometimes results in death as well. Actually the diagnosis of inflammatory processes relies on revealing anatomical/structural alterations of the affected organs and these changes are specific to the nature of the inflammation/infection under consideration. There is diversity in sensitivity, accuracy and specificity of various diagnostic techniques that mostly depends on the nature of the disease and pathophysiology operating there. The main objective of different imaging techniques is to incorporate the diagnostic functional data with that of anatomical/structural information in order to describe and characterize site, extent and activity of the disease [60].

After the development of various radiopharmaceuticals, the risk factors of morbidity or mortality accompanying infectious diseases have sharply decreased. ^99mTc based radiopharmaceuticals have a significant role in distinguishing infections from inflammations. Although scintigraphy images are based on functional abrasions of tissues even then inflammatory or infectious progressions can be visualized in their early phases, when anatomical alterations are not yet obvious.

Ciprofloxacin, a frequently used antibiotic is found to be active against most of the gram positive and gram negative bacteria, was labeled with ^99mTc. Ciprofloxacin is a fluoroquinolone-derivative antibiotic that binds to bacterial DNA gyrase and topoisomerase IV, and thus hinders the DNA replication [61]. The infections accurately detected by ^99mTc labeled ciprofloxacin (infector) are septic arthritis, prosthetic device infections, osteomyelitis, endocarditis, deep seated abscesses and extrapulmonary tuberculosis [62]. While ^99mTc-levofloxacin is found to be effective in diagnosing lungs, bone, sinus, airways, skin and joint infections, mostly caused by bacteria [63]. There are other fluoroquinolones derivatives also labeled which provide a higher labeling yield and better results than ciprofloxacin, viz. ^99mTc-clinafloxacin [64], ^99mTc-delafloxacin [65], ^99mTc-fleroxacin [66], ^99mTc-gemifloxacin [67], ^99mTc-norfloxacin [68], ^99mTc-rufloxacin [69] etc.

There are cephalosporins, antibiotics with greater effectiveness against gram-negative bacteria, ^99mTc-cefepime [70], ^99mTc-cefoperazone [26], ^99mTc-ceftizoxime [71], ^99mTc-ceftriaxone [72], ^99mTc-cefuroxime [73], etc. There also certain ^99mTc-antibiotics used to distinguish between sterile inflammation and bacterial infection namely ^99mTc-cefepime [70], ^99mTc-cefprozil [74], ^99mTc-clarithromycin [75]. Whereas ^99mTc-sulfadimidine can differentiate between septic and aseptic inflammation [76]. ^99mTc-daunorubicin, Mitomycin C and ^99mTc-doxorubicin are the only anticancer drugs which are also antibiotics, labeled with ^99mTc for brain imaging [77], liver imaging [16], and tumor detection [78] respectively. There are also many others given in the Table 1.

Table 1 ^99mTc-labeled antibiotics and antibiotic derivatives

^99mTc-labeled proteins, peptides, amino acids, and their derivatives

Amino acids are the building blocks of peptides and proteins, which are the major structural and functional units of living systems. Amino acids are present in the body and used in the metabolism. When there are higher energy needs, amino acids are also used for energy fulfilment. Thus in tumors, where there are neoplastic cells with high energy needs, ^99mTc-labeled amino acids or their derivatives prove to be good tumor locating agents. Some instances are ^99mTc-l-carnitine [11], ^99mTcN-PRODTC [79], ^99mTcN-PHEDTC and ^99mTcO-PHEDTC [80].

There are several receptors overexpressed in many types of tumors which can be bound by peptides. ^99mTc-labeled peptides, thus, are used as to target these overexpressed receptors and potentially better tumor localization [81]. Angiogenesis in tumors depends on the integrin α_vβ₃ expression which is overexpressed in various metastasizing cancers [82]. Several ^99mTc-labeled peptides have been synthesized which have high affinity to this receptor [83]. These include ^99mTc-RGD and its derivatives ^99mTc-[E-c(RGDfK)₂]₂ [83], ^99mTc-3P-RGD2 [84], ^99mTc-EDDA/HYNIC-RGD [85], given here within the Table 2. Other important receptors targeted for tumor imaging are somatostatin, GRP, EGF-R, and IGF-R, which are localized by ^99mTc-octreotide [86], ^99mTc-bombesin [87], ^99mTc-Ior egf/r3 [50], ^99mTc-Z_IGF1R:4551-GGGC [88] respectively, and their derivatives.

Table 2 ^99mTc labeled Proteins, peptides and amino acid derivatives

Immunoglobulins or antibodies are proteins synthesized by the body in response to some antigenic stimuli, which specifically bind to those antigens. So in the cases of tumor or infection, antibodies could be used to target the surface antigen of a cancerous cell, or antigens of the inflammatory agent, respectively. Monoclonal antibodies (Mabs) are the candidate for radiolabeling because they provide enough binding specificity for localization of the target. Certain ^99mTc-labeled Mabs are available for certain receptors or proteins, like ^99mTc-Ior egf/r3 [50], ^99mTc-D-AM-Fab and ^99mTc-D-HF [89]. There are other proteins that specifically bind to other proteins like affibodies. Affibodies are non-antibody low molecular weight proteins. ^99mTc-labeled affibody molecules serve as very good at localization of certain receptors overexpressed in tumors or other diseases, like ^99mTc-Z_IGF1R:4551-GGGC affibody which targets insulin growth factor type-1 receptors [88]. There are also many other protein and peptide derivatives labeled and given in the table 2.

Miscellaneous ^99mTc-labeled organic molecules

Miscellaneous macro and micro-organic compounds have been labeled with ^99mTc, as dictated by the nature and type of the ligand. Diphosphonates or bisphosphonates have affinities for bones. Many of their derivatives are labeled with ^99mTc in search of a superior bone scintigraphy imaging agent, e.g. ^99mTc-BIDP [90], ^99mTc-BIPeDP [91], ^99mTc-EIPrDP [47]. Nitroimidazoles have an affinity for hypoxic microenvironments of tumors, that’s why ^99mTc-nitroimidazole [92], and its derivatives e.g. 99mTc-metronidazole [22], ^99mTc-misonidazole [93], are used for tumor hypoxia imaging. There are other imidazoles like ^99mTc-omeprazole [94], ^99mTc-pantoprazole [95] and ^99mTc-rabeprazole [96] used for stomach ulcer localization. Also there is ^99mTc-tannic acid, one good radiotracer of stomach ulcer [49].

Our brain has billions of neurons which operate with nerve impulse. Nerve impulse is mediated as neurotransmitters bind to their receptors on the neuronal surface. Thus any ligand, might it be a neurotransmitter or its analog that can bind to these receptors, could be labeled for brain imaging. ^99mTc-amine-thiophene-dione [97], ^99mTc-gabapentin [98], ^99mTc-Histamine [99], ^99mTc-piracetam [100] are such examples. As the brain uses high energy for its functioning, glucose is a crucial requirement. Deoxyglucose derivatives are used for brain imaging like ^99mTc-ECB-DG [28]. While hepatobiliary imaging is facilitated by organic acids like ^99mTc-5-ALA [101], ^99mTc-BPIDA [102], ^99mTc-UDCA [103] etc. Cardiovascular diseases are detected and characterized by labeled β₁-receptor and other myocardial receptor antagonists. Labeled β₁-receptor antagonists are ^99mTc-labetalol [104] and ^99mTc-nebivolol. Another receptor angiotensin II is imaged by its ^99mTc-antagonist i.e. ^99mTc-losartan [105].

There are many ^99mTc-labeled ligand preparations that help to detect and characterize many types of tumors. They usually detect tumors by receptor-specific binding. For example ^99mTc(CO)₃-(1-azido-1-deoxy-β-d-glucopyranoside) for tumor detection [106], ^99mTc-clomiphene citrate is an estrogen receptor (ER) antagonist (breast and uterine cancers) [107], ^99mTc(CO)₃-folic acid derivative for folate receptors [108], ^99mTc-DES-P [109], ^99mTc-HYNIC-CHC for tubulin binding [110], ^99mTc-methotrexate (MTX) for folate receptors [31], ^99mTc-siRNA for chemokine receptor 4 expression [111] etc. Table 3 contains details of these and others. There are also non-antibiotic steroidal or non-steroidal compounds labeled with ^99mTc for infection or inflammation imaging like ^99mTc-2-aminoestrone-3-methyl ether [112], ^99mTc-celecoxib [113], ^99mTc-CSA-107 [114], ^99mTc-diclofenac [56].

Table 3 Miscellaneous ^99mTc-labeled organic molecules

Conclusion and perspectives

^99mTc labeled radiopharmaceuticals have an important place in medicine and health sciences. Because the role of ^99mTc in the diagnostics is very well established owing to its physical and chemical properties (half-life of 6 h, gamma ray energy of 140 keV, easily obtained from a ⁹⁹Mo/^99mTc generator, low cost, minimal dose to the patient and negligible environmental impact). High labeling yield with minimal harsh conditions like low specific activity, neutral pH etc. make a radiopharmaceutical best for practical applications.

By looking at the data presented here in this article, we can conclude that there are some superior diagnostic agents than the others. Superior ^99mTc-antibiotics include ^99mTc-benzyl penicillin, ^99mTc-cefepime, ^99mTc-cefuroxime axetil, ^99mTc-clarithromycin, ^99mTc-delafloxacin, ^99mTc-fleroxacin, ^99mTcN- GTND, ^99mTc-kanamycin, ^99mTcN-MXND, ^99mTc-pefloxacin, ^99mTc-rifampicin, ^99mTc-rufloxacin, ^99mTc-sitafloxacin, ^99mTcN-sitafloxacin dithiocarbamate (SFDE), ^99mTc(CO)₃-SFDE, and ^99mTc-temafloxacin complex (TMC).

Superior labeled proteins and peptide radiotracers include ^99mTc(CO)₃-hexapep, ^99mTc(CO)₃-tetrapep 1, ^99mTc(CO)₃-tetrapept 2, ^99mTc-(Nα-His)Ac-NT(8–13), ^99mTc-3P-RGD2, ^99mTc-anti-S. aureus antibody, ^99mTc-bombesin derivative (HYNIC-BB 5-14), ^99mTc-CpTT-octreotide, ^99mTc-Cys-annexin V, ^99mTc-EC-225, ^99mTc-ECDG ^99mTc-ghrelin peptide, ^99mTc-HYNIC-annexin A5, ^99mTc-HYNIC-GABA-Bombesin, ^99mTc-HYNIC-Tyr3-octreotide (TOC), ^99mTc-HYNIC-βAla-bombesin, ^99mTc-insulin complex, ^99mTc-Ior egf/r3, ^99mTc-neurotensin analog, ^99mTc-PADS, ^99mTc-trifoban, ^99mTc-trastuzumab, ^99mTc-ubiquicidin 29-41, ^99mTc-vasopressin and ^99mTc-Z_IGF1R:4551-GGGC.

Superior ^99mTc-labeled organic molecules from Table 3 include a heterogeneous group of compounds. ^99mTc-EIPrDP and ^99mTc-IPeDP are superior bone scintigraphy agents. Superior liver imaging agents include ^99mTc-5-ALA, ^99mTc-CMC, ^99mTc-Mitomycin C, ^99mTc-Vincristine and ^99mTc-UDCA. While a superior renal imaging agent is ^99mTc-DMSA. Some, very suitable, brain imaging agents are ^99mTc-amine-thiophene-dione, ^99mTc-BAT-AV-45, ^99mTc-histamine, ^99mTc-piracetam and ^99mTc-TRODAT-1. Some superior radiopharmaceuticals for gastrointestinal ulcer imaging are ^99mTc-famotidine, ^99mTc-omeprazole, ^99mTc-pantoprazole, ^99mTc-rabeprazole and ^99mTc-tannic acid.

Superior tumor imaging agents are ^99mTc-5FU, ^99mTc-DES-P and ^99mTc-methotrexate. While superior tumor hypoxia imaging agents are ^99mTc(CO)₃-4-nitroimidazole-triazole, ^99mTc(CO)₃-5-nitroimidazole-triazole, ^99mTc-HYNIC-MN, ^99mTc-misonidazole and ^99mTc-PyDA. Some very efficient infection/inflammation imaging agents are ^99mTc-2-aminoestrone-3-methyl ether, ^99mTc-celecoxib, ^99mTc-diclofenac, ^99mTc-piroxicam, and ^99mTc-fluconazole which is a fungal infection marker. Some superior and efficient heart imaging agents are ^99mTc-labetalol, ^99mTc-losartan, ^99mTc-nebivolol and ^99mTc-SestaMIBI.