Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours

Abstract

Targeted chemotherapy for cancer treatment offers a great potential advantage in tumour treatment due to greater specificity of delivery which leads to increased dose of the cytotoxin delivered to the tumour relative to the rest of the body. In order to achieve such selective targeted delivery one needs to identify generic markers that are over-expressed on the surface of tumour cells but are not over-expressed on normal tissue. Work of several authors has shown that some cells, such as those of rapidly dividing, aggressive tumours, over-express surface receptors involved in the uptake of vitamin B₁₂ [B. Rachmilewitz, M. Rachmilewitz, B. Moshkowitz, J. Gross, J. Lab. Clin. Med. 78 (1971) 275–279; B. Rachmilewitz, A. Sulkes, M. Rachmilewitz, A. Fuks, Israel J. Med. Sci. 17 (1981) 874–879] or folate [P. Garin-Chesa, I. Campbell, P.E. Saigo, J.L. Lewis Jr., L.J. Old, W.J. Rettig, Am. J. Pathol. 142 (1993) 557–567; O.C. Boerman, C.C. van Niekerk, K. Makkink, T.G.J.M. Hanselaar, P. Kenemans, L.G. Poels, Int. J. Gynecol. Pathol. 10 (1991) 15–25; G. Toffoli, C. Cernigoi, A. Russo, A. Gallo, M. Bagnoli, M. Boiocchi, Int. J. Cancer 74 (1997) 193–194; J.A. Reddy, D. Dean, M.D. Kennedy, P.S. Low, J. Pharm. Sci. 88 (1999) 1112–1118; J.A. Reddy, P.S. Low, Crit. Rev. Ther. Drug Carrier Syst. 15 (1998) 587–627; G.J. Russell-Jones, K. McTavish, J.F. McEwan, in: Proceedings of the 2nd International Symposium on Tumor Targeted Delivery Systems, 2002]. Furthermore the degree of over-expression has been found to correlate with the stage of tumour growth, with the highest levels found on stage IV carcinomas.

Using fluorescently-labelled polymers to which are linked the targeting agents, vitamin B₁₂, folate or biotin, the relative uptake of these polymers into various types of tumour cell lines grown both in vitro and in vivo has been examined. These studies have shown that while some tumour types do NOT over-express receptors involved in vitamin uptake, most tumour types over-express receptors for folate, or vitamin B₁₂. In either case there is also a greatly increased expression of a yet to be identified biotin receptor. In cases of receptor over-expression, binding of the targeted fluorochrome leads to rapid internalization of these molecules within the cells to levels that are two to thirty times higher than with non-targeted polymers. Using a number of cancer models, these studies were extended further and it was found that the increased expression of receptors also leads to increased levels of killing with targeted cytotoxins. Thus the preliminary data described suggests that the use of vitamins as targeting agents has enormous potential for use in cancer diagnosis and chemotherapy.

Introduction

In conventional cancer chemotherapy, in order to obtain a linear increase in killing of cancer cells (as the level of drug increases) it is often necessary to increase the amount of cytotoxic drug administered in an exponential fashion. This in turn leads to an undesirable increase in non-specific cytotoxicity of normal non-cancerous (by-stander), healthy cells. Because of this, normal cells such as hair, skin, and gut cells also die.

In order to reduce the undesirable side effects of cancer treatment it is often necessary to repeatedly deliver a smaller sub-optimal dose of the cytotoxin. However, this inevitably leads to the survival of a small fraction of drug-resistant cells. The most desirable cancer treatment, would, of course be one in which the anti-cancer agent targeted only tumour cells and did not enter non-cancerous normal tissue. In an attempt to increase the dose of cytotoxic agent delivered to the tumour cells, and reduce the level of drug going to by-stander cells, specific targeting agents such as monoclonal antibodies to “tumour-specific antigens” have been employed. In many cases, however, the antibody–drug conjugate used may be highly immunogenic, and thus lead to an antibody response against the conjugate, and thereby preclude its further use. Additionally these antibodies are generally specific for only a small number of tumour types. For this reason, small, poorly immunogenic, tumour-specific targeting molecules have been sought as alternatives to antibody molecules.

Whilst all living cells require vitamins for survival, rapidly dividing cells such as those present in solid tumour cancers have a voracious appetite for certain vitamins and as a consequence the receptors involved in uptake of the vitamins are over-expressed on these cancer cells. Accordingly, agents linked to certain types of vitamins should be highly attractive to tumour cells and should be useful in the imaging and identification of tumour cells. Additionally, cytotoxins linked to these agents should greatly aid in the treatment of tumours by delivering high doses of anti-cancer agents specifically to these cells. Recently focus has switched to the use of targeting molecules that are essential for growth of the tumour. Vitamin B₁₂, folic acid, biotin, and riboflavin are essential vitamins for the division of all cells, but more particularly for the growth of tumour cells. Considerable evidence has now accumulated which suggests that both vitamin B₁₂ and folate have potential use in targeting of imaging agents and pharmaceuticals to tumour cells.

There are existing anti-folate drugs which interfere with other parts of the folate cycle. These include chemotherapeutic drugs such as Methotrexate and 5-Fluoraracil (5-FU), which are widely used in cancer treatment. However their efficacy is limited by significant toxicity and ultimately the induction of drug resistance. Vitamin B₁₂ depletion has been demonstrated to kill cancer cells that are resistant to such conventional chemotherapeutic drugs.

Vitamin B₁₂ is an essential enzyme co-factor for methionine synthase (MS). This enzyme regulates one of the two major pathways for the production of folates, the cell’s primary carbon source for the synthesis of nucleotides and DNA. The anti-metabolite chemotherapeutic drug Methotrexate (MTX), inibits another critical enzyme of the folate cycle; Dihydrofolate Reductase (DHFR). Vitamin B₁₂ depletion, or exposure to MTX inhibits the production of nucleotide formation and DNA synthesis. Vitamin B₁₂ is acquired via the diet and is transferred across the digestive tract to the bloodstream where it binds with a carrier protein, transcobalamin II, (TCII). The B₁₂–TCII complex targets and binds to a specific high affinity receptor on the cell surface. The receptor is undetectable on non-dividing cells but is highly expressed when cells are stimulated to divide thus allowing for vitamin B₁₂ uptake. Rapidly dividing cells associated with cancers and other proliferative diseases express far more receptors than most normal cells. There is also some evidence of increased expression of the receptor on activated macrophages and some as yet unidentified cells in the lamina propria of the gut.

The cellular uptake of vitamin B₁₂ is facilitated by the plasma carrier protein, transcobalamin II (TCII), which is bound by a high affinity TCII receptor (megalin) located on the surface of the cell. Binding of TCII to the receptor is dependent on the formation of the VB₁₂–TCII complex (holo-TCII), as free TCII (apo-TCII) does not bind to the receptor [9], [10], [11] The Cbl–TCII complex is internalized by receptor-mediated endocytosis. The Cbl is then thought to be released from the TCII by the action of lysosomal enzymes. For functional activity, the vitamin B₁₂, which is bound to TCII is internalized by the cell. The vitamin must then be released from the internalized TCII receptor complex within the lysosome and then transported into the cytoplasm. The vitamin is then reduced from the +3 to the +2 state. It is then further modified to form methylcobalamin or adenosylcobalamin, which then binds to and activates either methionine synthase in the cytoplasm or methylmalonyl coA mutase in the mitochondria, respectively. Methylcobalamin is involved in the methyl transferase activity which converts methionine to homocysteine, following transfer of methyl group from N⁵-methyltetrahydrofolate, whilst 5′deoxyadenosyl-Cbl is the co-factor for molecular rearrangement of methylmalonyl-CoA to succinyl-coA (Fig. 1). In some tumour cells, such as the murine leukemia cell line, L-1210, the intracellular cobalamin concentration may reach 25 mM [12]. Rapidly dividing cells have a greatly increased demand for thymidine for incorporation into DNA and thus have high needs for cobalamin (Fig. 1). Hence, these cells increase their level of binding, uptake and storage of this vitamin. It is also possible that these rapidly dividing cells increase the level of synthesis of TCII, as it has been shown that the serum levels of ApoTCII (unsaturated TCII) are increased 3- to 26-fold in patients with acute promyelocytic leukemia [1], whilst increases have also been seen in patients with breast carcinoma [2]. The TCII receptor has been reported to be over-expressed in Leukemic cells, lymphoma cells, breast, lung, bone, thyroid, colon prostate and brain cancers and some other tumour lines.

Folic acid enters cells either through a carrier protein, termed the reduced folate carrier protein [13], or via receptor-mediated endocytosis facilitated by the folate receptor. The former receptor is of relatively low affinity and conjugates of drug to folate are not recognized by the reduced folate receptor, so drug–folate complexes are internalized via the folate receptor. There are two folate receptors FR-α, and FR-β. In general FR-α, is up regulated in malignant tissues of epithelial origin such as ovarian carcinoma (>90%) [3], [4] 122 of 136 epithelial ovarian cancers; [3], [14], lung (50%; [15]), breast (25%), endometrial, renal (50%), colorectal (including Caco-2 cells) [3], [7], while FR-β is over expressed in malignant tissues of non-epithelial origin (cancers of myeloid hematopoietic cells, brain (66% [16])) and placental cells [17], [18]. Interestingly the level of expression of the FR in tumours is directly correlated to the stage of the tumour [7]. Furthermore, Toffoli and co-workers [5] have suggested that the level of folate receptor expression in human ovarian cancers may be a predictor of a failure response to cis-platinum chemotherapy, with failures to therapy 14.1-fold higher in patients that had tumours with high FR levels. While the FR have been detected in normal tissues involved in the retention and uptake of the vitamin, these tissues are in protected sites and generally not accessible following blood-borne delivery of folate conjugates. Thus there is expression in the choroid plexus, the intestinal brush border apical membrane surface and the proximal tubules of the kidney. In the latter case the receptor probably functions to scavenge excreted folate, and as such would not be accessible to large molecular weight folate complexes.

Internalization of folate via the FR occurs following binding of folate to receptors clustered around caveolae. Large numbers of folate molecules can be internalized with over 60 million copies within 4 h in some tumour types [19]. Once internalized folate–BSA–gold nanoparticles were found to be internalized via calveolae, with subsequent transfer to multivesicular bodies, with association with the Golgi complexes and tubular endosomes [19], [20], [21].

Early work using monoclonal antibodies identified a tumour associated antigen, which was recognized by the monoclonal antibodies MOv18 and MOv19, which reacted with a surface antigen present on the majority of non-mucinous ovarian malignancies [22], [23], [5]. Subsequent work demonstrated that this protein was a glycolsylphosphatidylinositol-linked cell membrane protein with an apparent molecular weight of 38 kDa. Deglycosylation of the protein yielded a protein of 27 kDa [23]. Studies by Weitman and co-workers [16] demonstrated that this protein, termed GP38, was involved in the cellular accumulation of 5-methyltetrahydrofolic acid and was found on four of six brain tumors. This protein was subsequently identified as the high affinity folate binding protein, or folate receptor, and was also found to be recognized by another monoclonal antibody, LK26 [3].

One of the earliest reported examples of tumour imaging with vitamin B₁₂ was that of Flodh and co-workers [24], [25], [26]. These workers used ⁵⁸Co–VB₁₂ and ⁶⁰Co–VB₁₂ in autoradiographic studies of fibroblastic osteosarcomas, spontaneous mammary carcinomas, Ehlich ascities and Moloney virus-induced tumours in mice. These studies showed accumulation of label in the peripheral, actively growing tumours, with greatest accumulation in the sarcoma. Others tissues labeled included the kidney, gastric mucosa, reproductive and some endocrine organs. These studies are supported by the work of Wilbur and co-workers [27] who used radioiodinated arylstannylcobalamin conjugates and showed enhance uptake into renal carcinomas in nude mice when compared with muscle, intestines, spleen and lung. This work was supported by Warnock and colleagues [28] who observed increased uptake of ⁵⁷Co–VB₁₂ in sarcomas in mice when compared with gallium-67 and Thallium-201.

Collins and Hokenkamp [29] developed ¹¹¹In-diethylene-triaminepentaacetate (DTPA) analogs of VB₁₂ to image the CCL8 sarcoma in mice. In these studies they showed an increased half-life of the VB₁₂ analogue when compared to ¹¹¹In-DTPA, with much greater accumulation of the VB₁₂ analogue in the tumour than all other tissues apart from the kidney (cpm/gm).

These workers furthered their research into developing DTPA-adenosylcobalamin (DAC) as a cancer-detection compound, and found that it was effective in detecting high-grade aggressive tumours in humans [30], [31]. Some of the most promising results with the DAC compound were observed in a group of patients with breast cancer, where DAC successfully distinguished between malignant and benign tissue in eight of the nine patients, including one woman whose cancer was missed by physical exam, mammography and on an ultrasound scan. Other tumours such as high-grade metastatic breast, lung, colon, thyroid and sarcomatous malignancies were all successfully imaged, as well as central nervous system tumours and advanced metastatic prostate cancer. On the other hand low-grade malignancies and early skeletal metastatic disease were not effectively imaged. Despite this, the DAC compound was found to successfully image over 90% of tumours of the breast, lung, bone, thyroid, colon, prostate and brain using a standard gamma camera.

An alternative approach has been taken by Cannon and co-workers [32] who have synthesized fluorescently labeled cobalamin (Oregon green, naphthofluorescein and fluorescein) and used it to differentiate between neoplastic and healthy breast tissue grown in culture.

Early work on tumour diagnosis via the folate receptor relied on the use of the MOv18 and MOv19 monoclonal antibodies to identify the over-expressing cells in tumour sections, using FITC-conjugated anti-mouse antibodies [4], [5], [33], or alkaline phosphatase conjugated anti-mouse antibodies [16] amongst other techniques. The earliest example of in vivo imaging was with a folate-histamine derivative that contained ¹²⁵I [34] This analogue successfully imaged tumours in small animals, however the long half-life of the radionuclide (60 days) made it unsuitable for human use. More recently, Wang and Mathias [35], [36], [37] developed a deferoxamine-derivative of folate, which successfully chelated ⁶⁷Ga, however, this derivative still had an undesirably long half-life of 78 h. Even so it achieved a tumor-to-blood ratio of 409:1, 4 h after intravenous injection. Mathias and Green [37], [36], [38] developed a ¹¹¹In-DTPA–folate complex for imaging, which was used in phase I and II trials of patients who received 2 mg of the radiopharmaceutical containing 5 mCi of ¹¹¹In. SPECT (single photon-emission computerized tomography) images obtained with this substance demonstrated that the complex accumulated in the kidney and liver as well as in malignant, but not benign ovarian cancers [37], [39]. These studies have been extended by Leamon and Low amongst others [39] to the production of ^99mTc–folate conjugates with the hope of reducing the cost and half-life of the imaging agent with substantially reduced exposure of vital organs to the nuclide [40], [41], [42], [43]. Furthermore, the 140 keV γ-radiation emitted by ^99mTc is optimal for standard imaging equipment. Guo and co-workers synthesized a ^99mTC-6-hydrazinonicotinamido-hydrzido (HYNIC)–folate analogue which showed 300-fold higher uptake by tumour cells than non-specific binding. This analogue was also able to successfully image tumours of mice (24JK-FBP tumour) using gamma camera images.

In an alternative strategy Wiener and colleagues [44] produced 2-(4-isothiocyanatobenyl)-6-methyl-diethylenetriaminepentaacetic acid chelates which were also linked to folate and chelated to Gadolinium for use as imaging agents as well as FITC and TRITC-derivatized folate-dendrimers to produce both magnetic resonance imaging agents as well as fluorescent labeling reagents for tumour identification.

Two major limitations of the use of folate or vitamin B₁₂ in order to target tumor cells is that the dose deliverable is small, i.e., one molecule of drug for each molecule of targeting agent (TA), and that the majority of the TA–drug complexes are very small and as such are excreted by the kidneys and re-absorbed in the proximal tubules of the kidneys, thus leading to undesirable accumulation of TA–drug complexes in the kidney. This in turn results in damage to the kidneys, which is of course highly undesirable. Both of these disadvantages can potentially be overcome by the use of polymer-bound drug conjugates. The use of polymer-bound cytotoxins has the effect of increasing the dose of targeted drug delivery to the tumour as the increased molecular size of these conjugates stops them being secreted from the body through kidney filtration. Targeted polymer–drug complexes can also increase the amount of drug that can be taken into the cell due to targeting. Additionally, the use of targeted polymer–drug complexes makes use of an additional feature of tumour architecture, and that is the sporadic breakdown of the integrity of the vascular endothelium. This leads to the production of irregularly spaced holes in the endothelial layer allowing the extravasation of molecules up to 500 nm in size. Additionally, the lymphatic drainage within the tumour mass is often occluded, and hence large molecular weight molecules, such as polymers become “trapped” within the tumour mass. The combined result of these two phenomena leads to “Enhanced Permeability and Retention” (EPR) of high molecular weight molecules, and has been termed the EPR effect.