Keywords

32.1 Introduction

Cancer is complex yet commonplace, and the most terrifying disease of mankind. Among men in the USA, prostate cancer (PCa) is particularly lethal, second only to cancers of the lung and bronchus combined [1]. In 2020, more than 30,000 men will succumb to PCa, and more than 240,000 new PCa cases will be identified in the USA alone [1]. PCa affects one in every 6 men who are 60 years or older and affects African-Americans at a rate 2.4 times greater than European-Americans [1].

To treat these patients effectively, clinicians have a large array of options. However, even the novel agents such as abiraterone and enzalutamide offer only a limited survival benefit to the patients. For patients with skeletal metastases, FDA-approved Radium-223 chloride, (Xofigo) enhances the survival for up to 3.6 months. Furthermore Ra-223 chloride targets only osteoblastic lesions and does not provide effective treatment to visceral or nodal metastatic lesions. A continued search for more effective treatment has recently led to the development of a theranostic agent Lutetium-177-PSMA-617 (Lu-177-PSMA-617), although not yet approved by FDA for use in the USA, is being commonly used in many continents such as Europe, Asia, Australia and South Africa. At the time of this writing, the agent is in clinical trials in the USA. With three or more cycles of Lu-177-PSMA-617 treatments of approximately 7.4 GBq (200 mCi) each, progression-free survival of up to 13.6 months has been reported [2]. The best success of the Lu-177-PSMA-617 treatment consists of >50% PSA decline in 45% of the patients, partial regression in 56%, stable disease in 7% and progressive disease in 3.6% of the patients [2,3,4]. Progression-free survival for 3.6 months to 13.7 months has also been reported [2,3,4].

Although the results are encouraging, several weaknesses of Lu-177-PSMA-617 treatment have surfaced. First, PSMA is expressed only on 80% to 85% of PCa which requires patient to be screened, for PSMA expression using Ga-68-PSMA-11 PET scan. Second, extensive uptake of Lu-177-PSMA-617 in salivary glands leaves most patients with mild-to-severe xerostomia to minimize which some investigators have chosen to block the uptake using botulinum toxin (another pre-Lu-177-PSMA-617 treatment procedure). Third, considerable myelocytic toxicity and fatigue have been also reported in patients receiving Lu-177-PSMA-617. Fourth, Lu-177-PSMA-617 has approximately 75% renal excretion in 24 h, exerting radiation risk to renal medulla and bladder wall. Blocking renal uptake with certain amino acids prior to Lu-177-PSMA-617 treatment, a third pretreatment procedure, has been also considered. Fifth, Lu-177 contains approximately 0.1% Lu-177 m as an impurity. Lu-177 m has a half-life of 110 days. The urinary excretion of up to 75% of injected Lu-177 m within 24 h together with the longer lived Lu-177 m creates a waste disposal problem particularly for those patients who are incontinent and wear diapers.

These issues, in addition to the pretreatment procedures, not only add to the treatment cost but are also undesirable to the patients already stressed emotionally and weakened physically. In addition, the PSMA treatment eliminates 15 to 20% of the needy PCa patients who do not express PSMA. Improved agents with similar or better therapeutic effectiveness but without the persistent weaknesses are desirable.

32.2 Our Approach

Our proposed approach to treat PCa, its metastases and recurrence is driven by targeting an endogenous genetic product overexpressed when cells suffer genetic mutations that ignite cancerous transformation. VPAC mediates VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase activating peptide) growth hormone function in all types of PCa irrespective of the PCa heterogeneity [5,6,7,8,9].

These characteristic fingerprints, the VPAC cell surface receptors, express themselves at the onset of the malignancy, and may be prior to the elevation of PSA, and well before the cell morphology is altered this forms the basis of histologic diagnosis [5]. However, the expression on malignant cell surface has not yet been investigated for treating PCa and its metastases. Over the past few years, we have developed a small radioactive molecule (Cu-64-TP3805) that has high-affinity (Kd 3.1 × 10−9 M) VPAC receptors expressed in high density on all PCa cells. Our PET imaging studies in humans have shown that Cu-64-TP3805 detects primary PCa, bone metastatic lesions, and malignant lymph nodes with >95% sensitivity [10,11,12,13,14,15,16,17,18,19]. Furthermore, the agent has no urinary excretion, has no salivary gland uptake, no bone marrow uptake, and only a small uptake in the renal cortex, not sensitive to radiation damage.

Copper-64 has other radionuclide, a beta-emitting Copper-67, (t½ 2.6 day, γ-185 KeV (40%) and βmax- 580 KeV) which can be easily used to synthesize Cu-67-TP3805 using a well-established procedure in our laboratory and can be used for theranostic applications in PCa patients. Since (1) the tissue range (0.6 mm) and the linear energy transfer for Cu-67 is the same as Lu-177,and since Cu-67-TP3805, will have (2) the same tissue distribution as that of Cu-64-TP-3805 with high uptake both in primary PCa and its metastatic lesions, (3) no urinary excretion, (4) no salivary gland uptake, and (5) since VPAC is expressed in high density on all PCa types, the Cu-67-TP3805 treatment will be readily applicable to all PCa patients without having to perform patient suitability examination or having to deliver any of the antitoxicity, preventive procedures. These virtues of Cu-67-TP3805 for theranostic applications are listed in Table 32.1.

Table 32.1 Theranostic Cu-67-TP3805 at a glance
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32.3 VPAC Receptor and Its Expression on PCa

VPAC, a genomic biomarker, belongs to the superfamily of G protein-coupled surface receptors which are expressed in high density (104–105/cell) on all PCa types cells at the onset of oncogenesis [20,21,22,23,24,25]. On stroma, normal cells and benign masses VPAC is minimally present (5–10/cell). Since VPAC receptors are expressed on all PCa, for theranostic use of Cu-67-TP3805, patient-qualifying screening studies, like those required with Lu-177-PSMA-617 treatment, will not be necessary.

Reubi and colleagues [6,7,8,9] examined more than 600 tumors and their metastases using immunohistochemistry and conclusively reported that VPAC and VPAC2 receptors are overexpressed on a variety of frequently occurring human tumors including those of the breast and prostate. On 100% of the human prostate tumors examined (n = 35), VPAC receptors were predominantly overexpressed on PCa tissues and VPAC2 on stroma, to a lesser extent. Although VPAC receptors exist on normal cells, their expression is lower than on malignant cells on which the receptor density is high.

A 28 amino acid peptide VIP has high affinity for VIP receptors and the 27 amino acid peptide PACAP has high affinity for VIP and PACAP1 combined. VPAC receptors are overexpressed on all PCa including metastatic lesions (Figs. 32.1, 32.2, 32.3 and 32.4). High expression of VPAC receptors (>104/cell) has been observed by others. Both VIP28 and PACAP27 have high affinity for VPAC (VPAC and VPAC2 receptors) [6,7,8,9].

Fig. 32.1
2 scans. There is dark shade distribution in the head, stomach, and prostate regions in the Lu - 177 - P S M A, and the stomach region is highlighted in the Cu - 64 - T P 3805.

Comparison of tissue distribution of Lu-177-PSMA and Cu-64-TP3805 (targeting VPAC)

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Fig. 32.2
Two sets of P E T scans. Each set consists of 3 scans. The bright spot at the center marked by the arrow denotes the presence of tumor. The left scan represents Cu - 64 - T P 3805 and the right scan represents the radical prostatectomy.

PET images of two PCa patients (Gleason 3+4), 1 h post injection of approximately 4 mCi Cu-64-TP3805. Radical prostatectomy was performed 1 week later. Histology confirmed PCa malignancy

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Fig. 32.3
3 P E T scans. The first represents the Cu - 64 - T P 3805 P E T with bright spots throughout. The top right represents the Tc - 99 m M D P and the bright spots at the bottom scan represent the bone lesions. The zoomed image of both the lesions are marked on the left and the right.

A 70 year old male consented to the Cu-64-TP3805 PET imaging. Images showed multiple bone lesions secondary to his PCa. Histological examination of the bone biospy confirmed that the lesions were malignant

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Fig. 32.4
2 illustrations. A represents the P E T, P E T slash C T, and C T scan from left to right. The highlighted area marked by the arrow denotes the presence of the lesions. B denotes the lymph nodes for malignant tissue on the top and benign tissue at the bottom.

(a) Cu-64-TP3805 PET imaging of small lymph node (arrow, SUVmax 7.15) in a 61 y/o PCa patient. (b) Digital auto radiography (DAR) of a malignant lymph node (A). Left panel shows Cu-64-TP3805 bound to malignant cells as confirmed by histology (right panel) of the same lymph node. (B) Left panel benign lymph node has no Cu-64-TP3805 uptake. Histology (right panel) shows absence of malignant cells

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Therefore, we had hypothesized that radiolabeled VIP and PACAP1 or their analogues will provide us with excellent biomolecules for accurate and sensitive detection of human PCa. The probe can also detect metastases, and be used to determine therapeutic effectiveness.

32.4 VIP, PACAP, and Their Analogues

VIP is a 28-amino acid peptide initially isolated from porcine intestine [26]. VIP, whose structure is common in humans, pigs and rats, is a hydrophobic, basic peptide that contains three lysine (position 13, 18, 19) and two arginine (position 12, 14) residues. From the essential histidine residue at the N-terminus to the amidated C terminus, all 28 amino acids of VIP are required for high-affinity binding and biological activity [27].

VIP gene receptors (VIP1 and VIP2) have been detected on the cell membrane of normal intestinal [28] and bronchial epithelial cells [21,22,23] and are overexpressed on various cancer cells, including colonic adenocarcinoma [23, 29], pancreatic carcinoma [30], and cancers of the prostate [6,7,8,9]. VIP (Tyr10 and Tyr22) labeled with I-123 successfully imaged a number of human tumors [31]. Following PACAP homology, these gene receptors are recently named VPAC (for VIP1 and PACAP2 combined) and VPAC2 (for VIP2 and PACAP3 combined). PACAP, a 38-amino acid peptide, isolated from bovine hypothalamus, was named PACAP because it stimulated the accumulation of intracellular and extracellular cAMP in monolayer cultures of rat anterior pituitary cells [20, 32]. PACAP, a neurotransmitter and member of the VIP family, is ten times more potent than VIP in stimulating adenylate cyclase in pituitary cells [32]. PACAP has three gene receptors, PACAP1, 2, and 3. Gottschall et al. [33] isolated 27-amino acid PACAP (PACAP27) from bovine hypothalamus and concluded that PACAP38 and PACAP27 were equally active and derived from a single 176-amino acid precursor. PACAP27, like VIP, has an amidated C-terminus and histidine at the N-terminus. Nineteen of the 27 amino acids of PACAP27 are homologous. The fact that PACAP27 recognizes and has high affinity (Kd = 1.5 nM) for both VIP and PACAP (VPAC) receptors that are overexpressed on PCa cells suggests that PACAP or its bioactive analogue may also be a suitable agent to image PCa [8, 10,11,12,13,14,15,16,17].

32.5 Synthesis of N2(S-Benzyl)2 Containing VIP and PACAP

We synthesized one analogue of VIP28 (TP3939) and one of PACAP27 (TP3805) that are more potent and biologically stable than VIP28. Vasoactive intestinal peptide (VIP) bound to a C-terminal diaminodithiol (N2S2) chelator was synthesized on a Wang resin using ABI 341A peptide synthesizer (Applied Biosystems, Inc.) [10, 18]. The analogues were prepared, purified, and characterized by American Peptide Company (Sunnyvale, CA) and named after their molecular weights as TP3805 and TP3939. Although peptides have been conjugated with chelating agents such as DOTA (1, 4, 7, 10-tetraazadocdecane -N, N″, N′, N, −tetra acetic acid), it requires a prepared and pre-purified peptide to which DOTA is to be conjugated. The conjugated product then needs further purification and characterization. Preparation of our analogues is a one-step process that provides efficiency, saves time, and provides a N2S2 type of chelating moiety for strong chelation with Cu-64.

Our data show that these Cu-64 probes are highly stable in vivo [11, 12]. Furthermore, the high VIP affinity for receptors on malignant cells and subsequent internalization minimizes its proteolysis and allows cell detection, as we have demonstrated in both mice and humans [10,11,12,13,14,15,16,17,18,19, 34]. These analogues have the high IC50 values (4.4 nM and 5.3 nM, respectively) among the many that have been synthesized and evaluated [20, 32].

The rationale for choosing TP3939 analogues was as follows. VIP28 is comprised of three aromatic moieties at Phe6, Tyr10, and Tyr22, a negatively charged site at Asp3 and a lone pair structure at His1. Although all five sites are required for complete binding to receptors with high affinity, substitutions at position 22 of 3-OCH3–4-OH-Phe and Lys12, Nle17, Val26, Thr28-VIP produced the best results, increasing potency by four times (IC50 = 4.4 nM vs. 15 nM) over VIP28. Higher affinity may enhance tumor uptake and improve image quality. Again, our recent preliminary data in humans, obtained using Tc-99 m-TP3654, a VIP analogue are consistent with this hypothesis [34].

32.6 Cu-64-TP3805 and Its Tissue Distribution in Humans

Cu-64-TP3805, designed and extensively validated in our receptors laboratory, has a high affinity (3.1 × 10−9 M) for VPAC receptors [10, 18, 19]. The agent is highly stable in vivo, has no urinary excretion, has no salivary gland uptake, and has renal uptake only in the cortex, resistant to radiation damage (Fig. l). The hypothesis therefore is that by targeting VPAC receptors will eliminate (a) patient treatment qualifying PET imaging, (b) subsequent xerostomia without botulinum toxin pretreatment, and (c) reduce renal toxicity preventing renal pretreatment.

32.7 Ability of VPAC Target to Image Primary PCa, and Its Metastases in Bone and Lymph Nodes

Following targeting VPAC receptors, and validating our hypothesis in TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mice, we have studied 45 patients with primary PCa and metastatic lesions by PET imaging with Cu-64-TP3805 [18]. As confirmed by postsurgical histology, all primary and metastatic lesions were imaged with >95% sensitivity (Figs. 32.2, 32.3 and 32.4). These data support the notion that VPAC is a highly suitable target for theranostic applications of Cu-67-TP3805 for treating PCa and its metastatic lesions.

32.8 Suitability of Cu-67 for Theranostic Application

Copper-67 is a commercially available, 2.6 day half-lived radionuclide that has radiation characteristics similar to that of Lu-177 (Table 32.2), including its β tissue range of 0.6 mm and linear energy transfer (LET). Since Cu-67 has the same chemical properties as that of Cu-64, we can prepare Cu-67-TP3805 using the same well-established procedure in our laboratory. Therefore, Copper-67, available commercially without longer lived radionuclide contamination, can be easily prepared as Cu-67-TP3805 and be effectively used to target VPAC receptors. The attractive radiation characteristics of Cu-67 have already drawn considerable attention leading to clinical trials treating neuroblastoma using SARTATE™ composed of Cu-67-labeled peptide, MeCOSar-Tyr3-octreatate [35] (Table 32.2).

Table 32.2 How does copper-67 compare with Lu-177?
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Reflecting on the present, planning for the future and prompted by the highly encouraging results in our laboratory, our quest is to systematically investigate targeting VPAC receptors using beta-emitting Cu-67-TP3805 for theranostic applications of primary and metastatic PCa. In addition to the anticipated high benefit-to-risk ratio of Cu-67-TP3805 as a theranostic, VPAC receptors are expressed on many other oncologic diseases, such as the cancers of the breast, bladder, lung, ovary, and brain [6,7,8,9, 13, 17, 36, 37]. It is therefore, reasonable to postulate that Cu-67-TP3805 may serve as a useful theranostic agent to treat many other cancers as well.