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Targeting Chemotherapeutic Drugs to Prostate Cancer Cells Using Dendrimers Coupled to Anti-PSMA Antibody

Prostrate cancer is a serious and insidious disease. While improved screening techniques and hormone manipulation therapy have resulted in early diagnosis of prostate cancer and effective treatment for many men, this disease still impacts the male population significantly, accounting for approximately 38 percent of all deaths of men over the age of 50.

Importantly, despite the advances in diagnosis, there are real problems involved in therapeutic decisions. Early diagnosis has not clarified the benefits of aggressive therapy, such as radical prostatectomy, because the outcomes of this therapy in localized disease are not always curative. In particular, some patients with localized tumors will have progressive disease despite having prostate ablative therapy. The reasons for this are unclear but probably relate to differences in the cancer phenotype and the inability to identify minimally metastatic disease outside of the prostate at the time of prostatectomy.

Despite this, most men do well until their prostate cancer becomes resistant to hormonal therapy. When this occurs, there are few options because most chemotherapeutic protocols are ineffective and the disease is difficult to monitor. In particular, widely metastatic, hormone-resistant prostate cancer has an extremely poor prognosis. Thus, new therapeutic initiatives that would better characterize the extent of the disease and aid in the treatment of hormone-non-responsive prostate cancer would be extremely helpful.

We are developing targeting mechanisms for anti-cancer drugs directed towards PSMA-bearing cells that can subsequently be coupled to imaging and signal detection platforms. Through NIH-NCI grant support, we have developed a dendrimer-based platform for specifically targeting drugs, imaging agents and radiopharmaceutics to breast, head, and neck cancers. We are extending these studies to test anti-PSMA-dendrimer-drug conjugates for targeting of prostate cancer cells in tissue culture. We investigate the ability to target anti-PSMA antibody conjugates to prostate cancer cells in vivo and evaluate the ability of anti-cancer chemotherapeutic agents coupled to these conjugates to specifically kill prostate cancer cells in vivo.

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The work was started by Dr. James R. Baker and Dr. Anil Patri, a synthetic organic chemist, who has worked on the dendrimer design and construction and surface and internal modification of dendrimers, as well as on the loading of the dendrimers with humanized antibody to prostate-specific membrane antigen (PSMAext). They collaborate with Dr. Kenneth J. Pienta, who has extensive expertise in the area of prostate cancer, as well as Dr. Neil H. Bander from the New York Presbyterian Hospital and Cornell University Medical Center, who has worked on antibody-based targeting technology.  After Dr. Patri’s departure to NCI, Dr. Ramesh Shukla has been leading the chemistry work.

 

Technical Approach for Target Therapy: Smart, Nanomolecular Therapeutics
Given the requirements for designing a device small enough to efficiently enter cells but able to perform multiple smart tasks, the only currently available technology that serves these purposes is a nano-device. These are designed synthetic materials with structures less than 10 to 100 nanometers in size. Useful devices are limited to less than one hundred nanometers in diameter since this is the largest-sized particle that escapes the vasculature and that most cells will readily internalize. Unlike "nanomachines" based on fictional mechanical machines that have been "shrunken" to nanometer dimensions, several true molecular nano-structures have now been synthesized and used for drug delivery, gene transfer, antimicrobial therapeutics and immunodiagnostics. See "Dendritic Polymer Macromolecular Carriers for Drug Delivery" Anil K. Patri, István J. Majoros, James R. Baker Jr., Curr. Opin. Chem. Biol. 2002, 6, 466 as well as Patri AK, Kukowska–Latallo JF, Baker JR, Jr.: Targeted drug delivery with dendrimers: Comparison of the release kinetics of covalently conjugated drug and non covalent drug inclusion complex. Advanced Drug Delivery Reviews, 2005:57, 2203–2214.

Targeted Cancer Therapy
Targeted therapy is potentially an important means of attacking cancer. Better outcomes for hormone- resistant tumors may be achieved by specifically delivering drugs, radionuclides, or toxins to tumors to specifically kill tumor cells without affecting surrounding tissue. This is an attractive option for prostate cancer that is poorly responsive to standard therapy, particularly when the sensitivity of the cancer to an anticancer agent is similar to that of normal tissue. In addition, many types of cancer can infiltrate throughout normal tissue or bone and therefore cannot always be adequately addressed with external radiotherapy. See Shukla R, Thomas T, Peters J, Kotlyar A, Myc A, Baker JR, Jr: Tumor angiogenic vasculature targeting with PAMAM dendrimer-RGD conjugates. Chem Commun (Camb). 2005 Dec 14;(46):5739-41. Epub 2005 Oct 10.

This disease could also benefit from the targeting of radiopharmaceuticals or other agents specifically to the cancer cells. Targeted therapy is dependent on a specific marker for the cancer cells that can be identified with affinity agents such as antibodies (for antigens) or ligands (for receptors). Also, the coupling of imaging reagents to targeting agents can specifically identify cancer cells in a variety of tissues with greater sensitivity than traditional imaging.

Targeting in Prostate Cancer
In prostate cancer, the ability to target widely metastatic, hormone-resistant disease would be the most sought-after advance. This is because the control of non-hormone-responsive metastatic lesions would allow most men to live a nearly full life span and die for reasons other than prostate cancer since local disease rarely causes death. However, prostate cancer at this stage is resistant to most chemotherapeutic agents. Therefore, it would be of minimal help to merely target a drug to deliver higher concentrations to cells.

There may be advantages if the therapeutic could deliver the drug to specific parts of the cell, or maintain it within the cell to overcome the effects of multidrug resistance channels. Also, combinations of agents may yield synergistic anti-tumor effects. Targeted therapy with combinations of chemotherapeutic drugs may provide unique benefits in this regard because combinations of chemotherapeutic agents have shown utility in treating hormone-resistant prostate cancer, but this application is limited by toxicity.

Coupling dendrimers to these agents would not only target the drugs but could also solubilize some of these agents so they can be given intravenously. Additionally, the ability to target prostate cancer with combinations of radioisotopes and radio-sensitizing drugs could yield real benefits.

The combination of targeted therapeutics with targeted high-resolution imaging agents is also of potential interest. Current techniques of targeted imaging agents, using radio-scintillography, do not provide high-resolution images. Nonetheless, these techniques have been able to identify disseminated disease better than traditional imaging such as MRI or CT scanning. It is even possible that individuals considering local prostate ablative therapy or having metastatic disease could have therapeutic decisions modified on the basis of the finding of distant disease or could have adjunctive therapy that might lead to more durable response. Thus, targeted combination drug therapy for prostate cancer in conjunction with targeted imaging could yield true advances in care. 

Antibody-dendrimer conjugation

Scheme: Antibody-dendrimer conjugation Dendrimer modification.

The anti-PSMA antibody was conjugated to dendrimer by the following protocol:  To provide imaging capablity, the G5 PAMAM dendrimer 1 was treated with Fluorescene Isothiocyanate (FITC), purified by dialysis, and analyzed by 1H NMR, which showed characteristic aromatic signals for FITC conjugation.  This FITC modified PAMAM dendrimer 2 was reacted with a limited ratio of sulfosuccinimidyl 6-[3’-(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP), to introduce a disulfide group, to give 4.  The terminal amino groups of 4 were capped with excess acetic anhydride to give neutral surface dendrimer 5.  The disulfide group was cleaved with dithiothreitol to form the highly reactive thiolate 6. To provide a control, the FITC modified dendrimer conjugate 2 was capped with acetic anhydride to give 3.  All the conjugates were purified by dialysis, ultrafiltration and or gel filtration to remove excess reagent and small molecules, and characterized by 1H NMR and UV spectroscopy. Antibody conjugation. Modification on J591 anti-PSMA monoclonal antibody (MoAb):  To provide a thiol reactive maleimide group, the MoAb was reacted with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) in PBS at ambient temperature for 3 hrs. to give 8.  The unreacted reagent was removed on a PD-10 column.  The maleimide linked Antibody 8 was then reacted with dendrimer thiolate 6 in PBS-EDTA buffer to give the thioether cross linked dendrimer conjugate 9, which was quenched using N-ethylmaleimide to minimize dimer formation.  The product was ultrafiltered with microcon-100 to remove excess dendrimer and other reagents to give the desired conjugate 10.  It was further purified on FPLC using S-300 Sephacryl column.  This conjugate was analyzed using PAGE, and Spectrofluorometer to measure fluorescence.

Studies on the targeting of anti-PSMA antibody dendrimer conjugate in cultured cell lines (PSMA-positive LNCaP cells and PSMA-negative PC3 cells) have been performed and the results indicated that the conjugate targets LNCaP cells.  See "Antibody-dendrimer conjugates for targeted prostate cancer therapy" Anil K.Patri, Thommey Thomas, James R. Baker Jr., Neil H. Bander. Polym. Mater. Sci. Eng., 2002, 86, 130.

Targets in Prostate Cancer
Targeting agents associated with the prostate have been identified and characterized. At least two major prostate antigens have been identified: prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA). PSA was the first identified prostate antigen and has been remarkably useful in screening for this disease. However, there are several issues about using this as a target for therapy. First, it is secreted and present in high concentrations (micrograms/ml) in the serum. This could block targeting to tumor cells before the therapeutic or imaging agent can bind or enter a cancer cell. In addition, PSA appears to be less useful for targeting and is expressed at lower levels in hormone-resistant cancer. As a result, the ability to target hormone-non-responsive cancer, one of the major goals of targeting therapy, is less likely to be effective using PSA. In contrast, PSMA appears to offer theoretical benefits to targeted therapy and imaging.

PSMA is expressed by most prostate cancers and may also be expressed in the vasculature of prostate tumors. It is expressed at very high levels in most cancers, and it appears that expression of this antigen is maintained or may increase in hormone-resistant tumors or with hormone manipulation. Importantly, the levels of this antigen in serum are approximately 100-fold lower than that of PSA, suggesting that it would not be as likely to block targeting. 99Tc-labeled antibodies to PSMA have been able to identify prostate metastasis through imaging studies, and further characterization of the capability to identify metastasis may be achievable with higher-resolution imaging techniques. In addition, examination of lymph nodes of individuals undergoing radical prostatectomy suggests that the presence of PSMA (as detected by messenger RNA) is highly predictive of recurrence post-prostatectomy. Imaging approaches, however, are limited because of the inability to couple most agents other than radionucleotides directly to antibodies. Taken together, these findings suggest that PSMA may be a useful antigen to target therapy and imaging of prostate cancer. Therefore, the identification of metastatic disease and treatment of hormone-resistant prostate cancer are areas that potentially benefit from targeted therapy. See Patri AK, Myc A, Beals J, Thomas TP, Bander NH, Baker JR, Jr.: Synthesis and in vitro testing of J591 antibody–dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chemistry, 2004:15, 1174–1181.

J591 Anti—PSMA antibody—dendrimer conjugate for imaging and drug delivery

Figure 2: J591 Anti—PSMA antibody—dendrimer conjugate for imaging and drug delivery

PMPA-Dendrimer conjugates for targeting cells that overexpress PSMA.
Prostate specific membrane antigen (PSMA), an antigen on prostate cancer cells, also known as glutamate carboxy peptidase (GCPII), is over-expressed in prostate cancer cells, including difficult to treat metastatic hormone resistant cancers.  PSMA has been used for imaging of cancers and the literature shows that ligands against this antigen may be internalized into the prostate cancer cell. PSMA is highly structurally homologous to the neuropeptidase, NAALADase (N-acetylated alpha linked L-amino dipeptidase). Some analogs of 2-PMPA (2-Phosphonomethyl-pentanedioic Acid), a potent inhibitor of NAALADase activity, have been shown to bind and internalize into PSMA expressing prostate cancer cells.

Dr. Shukla uses Polyamidoamine (PAMAM) dendrimers as the drug delivery/release platform to which his team couples analogs of PMPA as targeting groups to construct a multifunctional, targeted drug delivery system for prostate cancer. They use Generation 5 PAMAM dendrimers as they are well suited for the development of a PMPA targeted drug delivery device and they are not likely to cross the blood brain barrier. Coupling these dendrimers to prostate targeting agents may not only target the drugs to localized as well as metastatic tumors, but could also give us the ability to target prostate cancer with combinations of chemotherapeutics and radio-sensitizing drugs that could yield significant benefits over existing therapies and imaging techniques. They are therefore developing a polyvalent drug delivery system for targeting prostate specific membrane antigen, using NAALADase inhibitor (2-PMPA) analogs attached to polyamidoamine (PAMAM) dendrimers.  Work on the synthesis, characterization and in vitro testing of PSMA targeting dendrimer-drug conjugates is in progress.

The Michigan Nanotechnology Institute is dedicated to the development of these devices. Our prostate cancer work has been funded by the Department of Defense (DOD)-U.S. Army Medical Research Acquisition Activity and the University of Michigan Comprehensive Cancer Center SPORE for prostate cancer.

 

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