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Improved Treatment of MT-3 Breast Cancer and Brain Metastases in a Mouse Xenograft by LRP-Targeted Oxaliplatin Liposomes

Orthmann, Andrea; Peiker, Lisa; Fichtner, Iduna; Hoffmann, Annika; Hilger, Ralf Axel; Zeisig, Reiner

 

Abstract:

The anti-cancer drug oxaliplatin (OxP) has rarely been used to treat breast carcinoma, as it cannot cross the BBB to treat the frequently subsequent brain metastases. Here, we encapsulated OxP in liposomes prepared to reduce side effects and to simultaneously treat primary tumor and brain metastasis. The angiopep LRP-receptor ligand was bound to the vesicular surface for targeting. Targeted and non-targeted OxP liposomes were tested in vitro (binding, uptake, and transcytosis) and in vivo. Liposomes contained 0.65 mg OxP/mL, their mean diameter was 165 nm, and they released 50% of OxP within 8 days at 4 °C and within 22 h at 36 °C. MDCK cells were used for uptake and transcytosis quantification. Compared to non-targeted liposomes, targeted liposomes showed 12-fold greater uptake, and 2.25-fold higher transcytosis. In vivo efficacy was tested using human MT-3 breast cancer cells transplanted subcutaneously and intracerebrally into female nude mice, and tumor growth inhibition was measured. OxP was injected (6 mg OxP/kg) four times. The best results were obtained with targeted liposomes (T/C: 21% for subcutaneous and 50% for intracerebral). OxP liposomes with a fluid membrane all inhibited MT-3 tumors significantly better than free OxP, with no significant difference between targeted and non-targeted liposomes. The therapeutic effect was accompanied with strong leukopenia and mild thrombocytopenia with all formulations. The newly developed OxP liposomes significantly improved the treatment of subcutaneously and intracerebrally growing breast cancer, but the targeted angiopep-equipped liposomes showed no superior effect in vivo.

Keywords: BLOOD-BRAIN-BARRIER; BRAIN TUMOR; LIPOSOME; OXALIPLATIN; TARGETING; XENOGRAFT

 

Supplement 

The remarkable progress in cancer treatment during last years, was mainly enabled by new biologicals and by personalization of treatment, but there is still a strong need for further improvement.

One possibility to further increase the efficiency of known anticancer drugs is a modification of the pharmacological profile by encapsulation, conjugation to an antibody (for targeted therapy) or both to increase the accumulation at target side and to reduce site effects.

Continuing our previous work using targeted liposomes to transport anticancer drugs across the blood brain barrier (BBB) [1-2] for the treatment of experimental brain metastases, we selected in the present study oxaliplatin (OxP) as anti-cancer drug to be encapsulated into ‘Trojan horse’ like liposomes. Many cytotoxic drugs are unable to efficiently cross the BBB because of their physicochemical properties, (polarity, lipophilicity, molecular weight and polar surface).

The therapeutic index of platinum compounds like OxP, but also cisplatin or carboplatin, commonly used for the treatment preferably of colorectal and lung cancers, is limited by nephrotoxicity, neurotoxicity or myelotoxicity and the short vascular circulation time [4].

In addition, these drugs are not able to cross the BBB. The BBB is a highly efficient morphological and physiological cell barrier formed by epithelial and endothelial cells. It separates blood vessels and surrounding brain tissue. The physiological barrier function of the BBB exerted by specific morphological and metabolic properties allows the selective exchange of essential nutrients to the brain and, on the other hand, protects it from harmful substances. The transport of ‘un-physiological’ material like drugs is therefore restricted and ‘tricks’ are needed to circumvent that border function to treat, for example, brain metastases. Brain metastases, which are frequently formed from primary carcinomas, usually result in a poor prognosis for the patient and effective treatments are lacking. It is assumed that 98% of all potential drugs for the treatment of brain metastases fail to overcome the BBB [3].

We suggest that our liposomal technology could extent the treatment opportunities of brain metastases.

We used MT-3/LS174T/LS174T cells as an appropriate model to investigate simultaneously both the growth of the primary, subcutaneously growing tumor and the ‘metastatic’ tumor, established by intracerebral transplantation into the brain.

Our idea was to encapsulate drugs into liposomes working like a ‘Trojan horse’ as well as to enable a targeting. Binding to the BBB is the precondition for a sufficient transcytosis from the blood into the brain (Fig. 1 scheme). We selected the LRP receptor, expressed at the surface of barrier forming cells, as target. It was shown, that a short 19mer peptide sequence (Angiopep), developed recently [5] bind efficiently to the LRP receptor and was used in our study as ligand.

In particular, we performed the following steps:

1. a) preparation of ligand baring and ligand free OxP-liposomes and their characterization
2. b) in vitro investigation to demonstrate transcytosis and cytotoxic efficiency
3. c) in vivo investigations concerning distribution and tumor growth inhibition by targeted therapy.

Vesicle preparation and characterization

OxP containing liposomes were prepared by reverse phase evaporation method, during which the drug was encapsulated. After separation of the non-encapsulated OxP by dialysis, the Angiopep ligand was conjugated to the drug containing liposomes by the post insertion method. Vesicles had a size of about 165 nm as determined by dynamic light scattering measurements. Total lipid concentration (26.1 – 37.9 mM), marker (74 – 83 mmol calcein/mol total lipid)) and OxP-concentration (up to 0.65 mg/ml) were determined by HPTLC, fluorescence measurements and HPLC, respectively. The shape of the vesicles was finally controlled by cryo-transmission electron microscopy (data not shown).

The liposomes were stable for more than 200 days, if stored at 4°C. The release of OxP was sufficiently fast with a release of 50% within 24 hours at physiological conditions.

 

In vitro experiments

Epithelial Madin-Darby canine kidney cells (MDCK) have been widely used to study transport processes across a tight cellular barrier and were utilized as a model to mimic the BBB. MDCK cells and human MT-3/LS174T/LS174T tumor cells were characterised for LRP receptor expression by flow cytometry. Both cells expressed the receptor, MDCK cells to a higher degree (~80% positive cells) than MT-3/LS174T cells (~50% positive cells), indicating that the target was sufficiently present for ligand-mediated transcytosis. An incubation of MDCK cells with targeted liposomes strongly enhanced uptake and transcytosis by about 300 and 220%, respectively, in comparison to non targeted, ligand free liposomes of the same composition.

Transcytosis experiments using a transwell chamber further demonstrated that about 50% of liposomal content was found insight MDCK cells after incubation for 24 h with liposomes loaded with the fluorescent dye calcein. In addition, the same amount of calcein was found in the basal media, demonstrating that about half of all liposomally encapsulated load could be transported across the tight cellular barrier.

In vitro cytotoxicity was assessed by the MTT proliferation test and revealed a high sensitivity of MT-3/LS174T/LS174T for OxP.

 

In vivo experiments

Pharmacokinetic (PK) parameters were determined, using female NMRI:nu/nu mice, transplanted with MT-3/LS174T tumor cells simultaneously at s.c. and intracerebral site. Mice were injected i.v. with a single dose of 10 mg OxP/kg given as free drug or encapsulated in non-targeted (nTL) and targeted, ligand baring liposomes (TL) and blood and tissue samples were taken at defined time points. These samples were analysed by ICP-MS measurements for their OxP content.

Free OxP had a distribution time C_max of 5.73 mg/l, while it was enhanced in a comparable way for targeted and non-targeted liposomes with C_max of 21.0 mg/l and 21.8 mg/l, respectively.

Accumulation of OxP in the brain was comparable for all three formulation with a peak concentration observed after 4 hours. (Fig. 2, C).

 

Inhibition of tumor growth was assessed using the same model, but with repeated treatments. Mice were stratified into experimental groups with 8 mice each when the tumor volume (TV) reached 0.1 cm³. Intravenous treatment with free and liposomal OxP in a dose of 6 mg/kg was performed at days 3, 7, 10 and 17 after tumor cell transplantation. This treatment schedule was well tolerated and was not accompanied by side effects like body weight reduction (Fig. 2 A).

 

Three days after first treatment and at the end of the study blood analysis was performed. Treatment with OxP in free or liposomal form always resulted in a strong leuko- and thrombocytopenia with a reduction down to 81% and 33% of control values, for red blood cells and leukocytes, respectively, indicating a certain amount of free OxP during circulation.

 

A significant growth inhibition of subcutaneously transplanted tumor was registered after therapy both with targeted and non-targeted OxP liposomes (Fig. 2 B) with an optimum T/C of 21.8% (day 19) and 47.9% (day 13), respectively.

Mice were sacrificed at day 19. Brains were isolated and snap-frozen. Cryo-sections were prepared and the tumor area was determined.

The therapeutic effect on brain tumor was different. While free OxP had no inhibitory effect, both liposomal formulations caused a significant inhibition with T/C values of 64% and 59%, respectively for non-targeted and targeted OxP-liposomes (Fig. 2 D).

 

These results indicate that the liposomes prepared in our lab are efficient transporters of the platinum compound OxP across the BBB to significantly inhibit brain tumor growth. The minor difference between the effect of targeted and non-targeted liposomes seems not only to be the result of the targeting alone but might also depend on the particular properties of the liposomes. We assume that especially the membrane fluidity of our liposomes contributed to the improved BBB transport and the high accumulation of OxP in the brain, responsible for the observed convincing tumor growth inhibition.

 

Importance of the study

Our results indicate that encapsulation of drugs into fluid membrane liposomes improves its therapeutic efficacy and enables the transport into the brain. It further extends the panel of tumor entities which can in this way be treated by drugs usually not belonging to the standard of care. It is suggested that this approach can be extended to other drugs.

Acknowledgement

This project was financed in part by a grant of the Federal Ministry of Economics and Technology of Germany, PRO INNO II (KA0453401UL7). The excellent technical support of Margit Lemm (MDC, Berlin-Buch) for preparing the tumor cells for in vivo experiments is gratefully acknowledged by the authors.

 

Abbreviations:   TL: targeted OxP-liposomes, nTL: non-targeted OxP liposomes, OxP: Oxaliplatin (in free form)

 

References

1       A. Orthmann, R. Zeisig, T. Koklic, M. Sentjurc, B. Wiesner, M. Lemm, and I. Fichtner, Impact of membrane properties on uptake and transcytosis of colloidal nanocarriers across an epithelial cell barrier model, J. Pharm. Sci., 99 (2010) 2423-2433.

2       A. Orthmann, R. Zeisig, R. Süss, D. Lorenz, M. Lemm, and I. Fichtner, Treatment of experimental brain metastasis with MTO-liposomes: impact of fluidity and LRP-targeting on the therapeutic result, Pharm. Res., 29 (2012) 1949-1959.

3       A. Orthmann, I. Fichtner, and R. Zeisig, Improving the transport of chemotherapeutic drugs across the blood-brain barrier, Expert. Rev. Clin. Pharmacol., 4 (2011) 477-490.

4       Galanski M. Recent developments in the field of anticancer platinum complexes. Recent Pat Anticancer Drug Discov. (2006) 285-95.

5       M. Demeule, A. Regina, C. Che, J. Poirier, T. Nguyen, R. Gabathuler, J. P. Castaigne, and R. Beliveau, Identification and design of peptides as a new drug delivery system for the brain, J. Pharmacol. Exp. Ther., 324 (2008) 1064-1072.