Polymeric Drug Delivery I - American Chemical Society

Chapter 20

Chelants for Delivery of Metal Ions

Downloaded by STANFORD UNIV GREEN LIBR on July 7, 2012 | http://pubs.acs.org Publication Date: February 16, 2006 | doi: 10.1021/bk-2006-0923.ch020

R. Keith Frank*, Philip S. Athey, Gyongyi Gulyas, Garry E. Kiefer, Kenneth McMillan, and Jaime Simón The Dow Chemical Company, Freeport, TX 77541 *Corresponding author: [email protected]

Chelating agents play an important role in the delivery of metal ions for medical applications. These applications in clude both diagnosis and therapy. In diagnostic applications, the metal ions can be radioactive (gamma-emitter), para magnetic, or fluorescent. Therapeutic applications generally require metals that are either chemically active on their own or emit ionizing radiation (beta or alpha-emitter). The ability of the chelant to form a stable in vivo complex is critical to its role in delivering the metal ion effectively and safely to its target. Examples using chelating agents for metal ion delivery in medical applications include therapeutic bone agents and bifunctional chelating agents.

Introduction Chelating agents play an important role in the delivery of metal ions for medical applications. These applications include both diagnosis and therapy. In diagnostic applications, the metal ions can be radioactive, paramagnetic, or fluorescent. Radioactive metals such as technetium-99m ( Tc) are used in nuclear medicine procedures to evaluate physiologic conditions such as bone cancer, 99m

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© 2006 American Chemical Society

In Polymeric Drug Delivery I; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


283 renal function, or heart disease. In these applications, y-emitting isotopes are used in conjunction with either simple scintillation cameras or more complex imagers such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). Very precise anatomical information can be ascertained by magnetic resonance imaging (MRI). A contrast agent con sisting of chelated paramagnetic metal ions such as gadolinium (Gd) are typi cally used to enhance images in MRI. Therapeutic applications generally require metals that are either chemically active on their own or emit ionizing radiation. Ionizing radiation used in thera py is typically particulate (P or a), but photon (y) emissions can be used as well. Therapeutic effect is achieved with the cell-killing caused by hydroxyl radicals formed by the interaction of ionizing radiation with water. Occasionally, metal ions can be administered as a simple salt, as is the case for Metastron® ( Sr chloride) for bone pain due to metastatic bone cancer. More commonly, however, a chelating agent is required to control the bio distribution and pharmacokinetics of the metal ion. Sometimes a metal ion is conjugated to a carrier moiety using a bifunctional chelant, depicted as the claw in Figure 1. The carrier may be a polymer or a biological targeting molecule such as a monoclonal antibody (mAb) used in radioimmunotherapy (RIT). 89

Figure 1. Roll of a bifunctional chelant in a conjugate. (See page 5 of color inserts.)

The ability of the chelant to form a stable metal ion complex is critical to its role in delivering the metal effectively and safely to its target. Therapeutic bone agents and bifunctional chelants are two example chelating ^gent applications for metal ion delivery that will be discussed.


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Results and Discussion Therapeutic Bone Agents Quadramet® (Samarium Sm-153 Lexidronam Injection) The bone-seeking chelating agent Lexidronam, also known as ethylenediaminetetramethylenephosphonic acid (EDTMP), has been used to deliver the P-emitting radioisotope Sm to sites of bone lesions. The therapeutic bone agent Quadramet, shown in Figure 2 (without cations and waters of hydration), was approved in 1997 for the treatment of bone pain due to metastatic bone cancer.


,53

Figure 2.

Sm-EDTMP (Quadramet ) for bone cancer. ( Cations and waters of hydration not shown.)

,53

Sm is a moderate-energy p emitter (702 KeV - 44.1%, 632 KeV - 34.1%, 805 KeV - 21%) but also has an imagable y photon (103 KeV - 28.3%), which allows viewing the biolocalization of the Sm using a scintillation camera, as shown in Figure 3. There is a lack of any accumulation in soft tissue with only skeletal uptake evident. In general, about half of the injected radioactivity accumulates in the bone with the remainder being excreted very rapidly via the kidneys (less than two hours in rodents). I53



285

Figure 3. Sm-EDTMP scintillation scan 3 hours post injection in a rabbit. (See page 6 of color inserts.) One of the benefits of bone-seeking radiopharmaceuticals such as Quadra met is that they have higher uptake in lesions or rapidly growing bone than in normal bone. This behavior is illustrated in the rabbit drill-hole model shown in Figure 4. In this model, small holes are drilled in the femur to simulate a bone tumor. After injection of Sm-EDTMP, the rabbit is imaged using a scintil lation camera, as shown on the left. On the right is an image of the lesion, showing a 20-fold excess of radioactivity in the lesion versus the surrounding normal bone. 153

Figure 4. Rabbit drill-hole model. Left - scintillation camera. Right - image shows selective uptake of Sm-EDTMP in drill-hole lesion. (See page 6 of color inserts.) 153


286 153


Figure 5 shows an image from an early human trial of Sm-EDTMP. Sites of metastatic bone disease are clearly visualized by the selective uptake of the radiopharmaceutical.

153

Figure 5. Image from human trial of Sm-EDTMP showing sites of metastatic bone tumors. (See page 6 of color inserts.)

EDTMP forms a moderately labile complex with Sm in vivo. A large ex cess of EDTMP over Sm (nearly 300-fold) is required in order to control the delivery of the metal ion and prevent uptake in non-target tissues. At much lower chelant-to-metal ratios, significant amounts of radiometal accumulate in tissues such as the liver. For example, as can be seen from the data in Table , the uptake (percent injected dose) in the liver increases as the EDTMP to Sm ratio decreases (I).

Table I. Liver uptake of

Sm in rats at 2 hours with varying EDTMP to Sm ratios.

EDTMP:Sm ratio

Liver (%ID)

38 76 115 153 268

0.79 0.59 0.37 0.33 0.25


287 166

Skeletal Targeted Radiotherapy ( Ho-DOTMP) 153

A developmental spin-off of Sm-EDTMP is Skeletal Targeted Radio therapy (STR™). STR is currently in late stage clinical evaluation for bone marrow ablation as part of a bone marrow transplantation protocol for the treatment of multiple myeloma. For this application, the bone seeking chelant, 1,4,7,10-tetraazadodecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP), is being used to deliver the p-emitter Ho (Figure 6) to bone.


l66

o

o

o

o

,66

Figure 6. Ho-DOTMP (STR) for bone marrow ablation. (Cations and waters of hydration not shown.)

,66

Ho was selected because of its high P energy (1854 KeV - 51%, 1774 KeV - 48%), which allows complete ablation of the bone marrow to be achieved. Because of its short half life (26.8 hours), the actual transplant can occur within a few days. This is important because patients having undergone bone marrow ablation are immune-compromised and prone to infection. Ho also has an imagable y photon (81 KeV - 6.2%), which allows for visualization. The macrocyclic DOTMP chelant forms very stable lanthanide complexes. Due to this high stability, a large excess of chelant is not required. This is important in this application because, in order to achieve complete ablation, a large amount of radioactivity is required. It is undesirable to inject a large amount of free chelating agent due to potential toxicity concerns. l66


288 Bifunctional Chelating Agents Bifunctional chelants (BFC) are molecules that have the capacity to sequester a metal ion and, in addition, have the facility to become attached to another entity. The three parts of a BFC molecule are illustrated below in Figure 7. Shown is the BFC a-[2-(4-nitrophenyl)ethyl]-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (PA-DOTA). There is a chelant portion, a linker portion, and a spacer connecting the two. The chelant portion of the BFC binds the metal ion and, for (+3)-metals such as Y , 'in, or Lu, is typically either an acyclic diethylenetriaminetetraacetic acid (DTPA) analog or a macrocyclic l,4,7,10-tetraazacyclodocane-l,4,7,10-tetraacetic acid (DOTA) analog (shown). The linker is a reactive group that can be attached to a carrier. Shown is a typical isothiocyanate, which can be used to attach to lysine e-amines on a protein. Various spacing groups have been used to alter the biological proper ties of the final conjugate.


90

11

C0 H

H0 C

2

2

^ ^ y - " Linker Spacer

177

v

'

Chelant

Figure 7. PA-DOTA bifunctional chelant showing the three parts of the BFC.

Biological Targeting Molecules Biological targeting molecules such as monoclonal antibodies (mAb) can be conjugated to BFCs to deliver metal ions to specific sites in the body. This has both diagnostic and therapeutic applications. For example, a cancer-seeking mAb can be labeled with a radiometal to visualize sites of metastatic disease. In radioimmunotherapy, this radiolabeling combines the cytotoxic nature of ionizing radiation with the targeting ability of mAbs to destroy cancer cells. An example of such a biotargeted radiopharmaceutical is Zevalin™, developed by IDEC and recently approved as a treatment for non-Hodgkin's lymphoma.


289 It is particularly important in therapeutic applications that a stable BFCmetal ion complex be maintained in vivo. Failure to achieve suitable stability will allow the radioactive metal to be released and thus accumulate in non-target tissue. This can be illustrated by an experiment comparing the three bioconjugates shown in Figure 8. These identical CC49 F(ab') fragments were conjugated to three different BFCs, Bz-DTPA, MX-DTPA, and PA-DOTA. They were all labeled with L u and injected into BALB/C mice (n=5). The percent injected dose (%ID) in the liver over time post-injection (PI) is shown in Figure 9. 2


177

Bz-DTPA II

C0 H C0 H 2

2

Me /—C0 H 2

N. N H ^ N F T H

^

f

i

^CO H 2

C

^]

MX-DTPA

°2H

C0 H CO H 2

2

PA-DOTA

Figure 8. Three CC49 F(ab') bioconjugates based on Bz-DTPA, MX-DTPA, and PA-DOTA. 2

The three bioconjugates show significantly different uptakes of Lu in the liver. These are inversely related to the stability of the metal complexes (i.e.,



290

Bz-DTPA forms the least stable Lu-complex and results in the highest Lu liver uptake, whereas PA-DOTA, which forms the most stable Lu-complex gives the lowest Lu liver uptake). Similar biodistribution differences were seen in the bone (not shown). The increased localization of Lu in these tissues for less stable complexes can be explained by radiometal "leaking" out of the chelant. It is well know that "free" lanthanides such as Lu will accumulate in the liver and bone. Free metal ions form colloids which go to the liver, and lanthanides are calcium ion mimics in the bone. Calcium mimicking by lanthanides is clearly undesirable, as accumulation in non-target tissues such as liver or bone can result in undesired radiotoxicity. New BFCs such as a-(5-isothiocyanato-2-methoxyphenyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (MeO-DOTA), shown in Figure 10, have been specifically designed to maintain stable in vivo complexes, particularly for lanthanide metal ions. 177


291

C0 H


2

C0 H 2

Figure 10. MeO-DOTA bifunctional chelating agent.

Nanoparticles Nanoparticles are of great interest because they have the potential to carry a large payload. BFCs are being used to attach metal ions, particularly Gd, to nanoparticles. Gd, being a paramagnetic metal, is important as a contrast agent in magnetic resonance imaging (MRI). Unlike radioisotopes, however, a relatively large amount of Gd is required for imaging. Whereas a single isotope attached to a biological targeting molecule (e.g. an In-labeled mAb such as ProstaScint®) could make an effective imaging agent, this is not the case with Gd. Nanoparticles allow a large number of Gd ions to be localized at target sites. nl

Carbohydrate Hydrogel Nanoparticles Carbohydrate hydrogel nanoparticles are one example of this kind of system. Targeting peptides such as integrins, EGFR, and VEGR-2 can be attached to the surface in order to target the nanoparticle to particular sites (2). The Gd-chelate of MeO-DOTA, shown on the left in Figure 11, has been used to bind multiple Gd ions to the nanoparticle (not shown in the figure is the coordinated water molecule essential for MRI contrast). The isothiocyanate is used to react with amine groups on the particle surface to form a covalent linkage. A depiction of the targeted nanoparticle is shown in Figure 11 on the right.



292

Figure 11. Gd-MeO-DOTA bifunctional chelate (left, shown without water of hydration) and depiction of its use in a targeting nanoparticle.

Lipid Nanoparticles Another type of particle being developed is lipid-covered perfluorocarbon nanoparticles containing targeting molecules on their surface. The targeting molecules enable the particles to accumulate in certain sites such as arterial plaque. One goal of tins work is the targeted delivery of a contrast agent for MRI (3). BFCs have been modified to contain lipophilic tails, designed to incor porate themselves into these lipid nanoparticles. Compounds such as DTPA-PE, shown in Figure 12, are being evaluated as a means to attach a large number of Gd-ions to targeted lipid nanoparticles (4).

o

CH —(CH2>14 3

2

O—

CH —(CH )i4 3

C0 H

ii

2

O

OH '—O—P—o,

II

o

Figure 12. Chemical structure of DTPA-PE.


C0 H 2

C0 H 2

293 An example of a targeting lipid nanoparticle is represented in Figure 13. The BFC shown is a derivative of MeO-DOTA and has a lipophilic tail, which allows it to be attach to the lipid monolayer. If the metal ion " M " in the figure is Gd, one can make an MRI contrast agent. A radiopharmaceutical can be produced if " M " is a radiometal. This could be diagnostic, such as In or a therapeutic such as L u or Y . 111


177

9 0

Figure 13. Targeting lipid nanoparticle. (See page 7 of color inserts.)

Conclusions In summary, chelating agents can be used to effectively control the destiny of metal ions in the body. The chelating agent is important, and not all chelants are created equal. Looking toward the future, medical chelants for metal ion delivery will continue to evolve. Next generation chelants will have varying charge and/or lipophilicity, which can provide improved pharmacokinetics of small molecules such as targeting peptides. A variety of linking groups and methodologies will provide conjugation under very mild conditions and will provide chelants suitable for use in solid-phase peptide synthesis. Novel chelant designs will allow for faster metal complexation kinetics while maintaining in vivo stability.


294

References


1. Unpublished data from The Dow Chemical Company 2. Unpublished information from Alnis BioSciences, Inc. 3. Wickline, S.A.; Lanza, G.M. Nanotechnology for molecular imaging and targeted therapy. Circulation 2003, 107, 1092-1095. 4. Winter, P.M.; Caruthers, S.D.; Yu, X.; Song, S.K.; Chen, J.; Miller, B.; Bulte, J.W.M.; Robertson, J.D.; Gaffney, P.J.; Wickline, S.A.; Lanza, G.M. Improved molecular imaging contrast agent for detection of human thrombus. Mag. Reson. Medicine 2003, 50, 411-416.


Polymeric Drug Delivery I - American Chemical Society

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