Multidrug resistant (MDR) cancer and circumvention thereof

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Multidrug resistant (MDR) cancer and circumvention thereof

Nearly half of all patients with cancer suffer from malignancies that are intrinsically resistant to chemotherapeutics. Furthermore, the majority of the remaining half will acquire resistance during the course of their therapy. [5] MDR has been described as the “thorniest obstacle” in developing improved systemic therapies for disseminated cancer. [6] Resistance to various structurally and mechanistically unrelated chemotherapeutics characterizes the MDR phenotype. Categorically, MDR is classically associated with the over-expression of ATP binding cassette (ABC) transmembrane efflux pumps, particularly P-glycoprotein (Pgp). Pgp is a 170 kDa transmembrane “permeability” glycoprotein. Among other ABC transporters, multidrug resistance associated protein (MRP) and breast cancer resistant protein (BCRP) are also of clinical relevance. [7] These transmembrane proteins are energy dependent pumps that can efficiently expel various structurally and mechanistically unrelated chemotherapeutics including Epipodophyllotoxins (etoposide), Taxanes (paclitaxel) and Vinca alkaloids (vinblastine) to the extracellular environment (Figure 2.1.) thereby maintaining intracellular concentrations below effective cytotoxic levels. [8] Increased expression of these multidrug resistance proteins is associated with a poor response to treatment and grave prognosis. Numerous different compounds have been shown to inhibit the efflux activity of Pgp and other ABC transporters thus restoring sensitivity to cytotoxic agents. [9,10] Unfortunately, due in the main to toxicity and poor pharmacokinetic control (altered distribution and thus altered toxicity profile), no such agents (termed chemosensitizers) are yet clinically available. [11] Verapamil and cyclosporin A are two examples of first generation chemosensitizers. Second and third generation chemosensitizers were developed with the aim of achieving more specific Pgp inhibition and fewer systemic pharmacological effects.

Combination chemotherapy

Combination chemotherapy has been the mainstay of successful (curative) chemotherapy for the last 40 years, [12, 13] so much so, that it is now considered standard practice. [14] A strong correlation exists between the number of agents administered and successful cure rates. [15] Combination chemotherapy regimens have predominately been developed empirically in late-stage clinical trials. Active single agents have typically been combined on the basis of minimizing the potential for overlapping toxicities (different organ toxicities) and possessing different (hopefully complementary), non-cross-resistant mechanisms of action. [16] These poly-chemotherapy regimens offer the potential for increased efficacy (greater fractional affect), increased neoplastic specificity, decreased dosage (hence reduction in systemic toxicity) and a broader spectrum of activity against sub-populations present within a heterogeneous tumour cell population which in turn minimises the development of resistance. [12] Clinically, the drugs are typically administered at their individual maximum tolerated doses. [14] This “more-is-better” approach ignores the possibility of subtle concentration and ratio dependent interactions capable of eliciting synergistic responses. [17, 18] Fixed-ratio drug combinations (FRDC) have recently been proposed as a more rational approach for the combination of drugs. Through in vitro optimisation, FRDC hold the promise of capturing maximal synergistic drug interactions thus reducing the total dose required to produce a particular fractional affect.

Tetramethylpiperidine (TMP)-substituted Riminophenazines

Over the course of the last 40 years, hundreds of analogues have been synthesized allowing for extensive quantitative structure activity relationships (QSAR) to be performed. Modifications have focused on substitution in the imino nitrogen region of the molecule (at position 2 of the phenazine nucleus) along with varying halogenation profiles in the phenyl- and anilino-rings. Tetramethylpiperidine substitution (Figure 2.2.) at the imino nitrogen position in comparison to the isopropyl group found in clofazimine has been shown in vitro to incur superior direct cytotoxicity against several intrinsically resistant neoplastic cell lines. [35] In addition they have been shown to possess greater chemosensitization activity using various Pgp expressing cell lines. [25, 37, 38] These Riminophenazines thus possess the potential for inclusion in several chemotherapeutic regimes. B4125 has been identified from a review of the structure activity relationships (Appendix B) as possessing the best neoplasticspecific cytotoxicity.

Loco-regional chemotherapy

Although systemic chemotherapy has proved effective in certain haematological malignancies (Acute myelogenous leukaemia) and a few solid tumours (notably testicular cancer), to date there is relatively poor efficacy against most systemic therapies (due to a lack of targeting) and the vast majority of dessimanted cancers remain incurable. Since Klopp et al. [40] first attempted intra-arterial chemotherapy in 1950 there has been a growing interest in delivering cytotoxic agents locally into the region of tumour growth via the artery supplying the region. The rationale being to expose the tumour to higher drug concentrations (above the maximum tolerated dose) thus producing a greater fractional tumour cell kill whilst limiting the side effects as systemic exposure is reduced. Regional delivery is thus an approach used to increase the exposure of cancer cells to drug/s beyond what can be achieved safely through systemic drug delivery. [41] Regional chemotherapy can be divided into two categories: third space regional compartment therapy (e.g. cerebrospinal fluid space, peritoneal cavity, pleural space etc.) and intra-arterial infusion into an afferent artery feeding the tumour containing organ or body region.

The Enhanced Permeability and Retention (EPR) effect

The enhanced permeability and retention effect (Figure 2.3.) of macromolecules and lipid-based particles is a general characteristic of viable and rapidly growing solid tumours. The increased vascular permeability underlying this effect is often considered the other side of the angiogenesis coin. [48] The EPR effect has been described both as the “gold standard” [48] and as the “royal gateway” in the design of new anticancer agents. [49] Selective, passive targeting via the tumour vasculature is possible as result of extensive production of vascular permeability mediators including bradykinin, nitric oxide, vascular endothelial growth factor, peroxynitrite, prostaglandins and matrix metalloproteinases that promote angiogenesis or facilitate extravasation. Defective tumour vasculature that lack a continuous smooth muscle layer and contain gaps in endothelial cell-cell junctions further contribute towards permeability. This results in leakage of colloidal blood plasma components such as polymer conjugates and nanoassemblies (typically between 10 and 500 nm, dependent upon model) into the tumour tissue.

Intelligent formulations: Nanoparticulate drug delivery systems

Drugs are inanimate, exogenous chemical entities that have affinity for and intrinsic activity upon a receptor that can then elicit a biological response. The chemical structure of a drug dictates its pharmacological utility (both dynamic and kinetic). The physicochemical properties of a drug determines to a large extent, the ability of a drug to be adequately absorbed and distributed to the intended biophase. [69] With the advent of combinatorial chemistry, computational in silico modelling, genomics and proteomics (supplying targets) there is an abundance of novel chemical entities that are emerging as drug candidates. Invariable in vitro assays are the means by which a particular target or suspected activity is determined (validated) using multi-well bioassays. As such, molecules with potent pharmacodynamics are discovered in high throughput screening (HTS) without Chapter 2: Literature study 18 much consideration for their pharmacokinetic properties. Possessing impressive pharmacodynamics does not mean impressive pharmacokinetics.

Pre-clinical development of anticancer drug products:

Regulatory perspectives Pre-clinical development encompasses all the activities required (deemed necessary to ensure safety) before a new chemical entity can enter into human trials. The goal of pre-clinical studies is to provide accurate, reliable and timely data that will be used to justify the conduct of clinical trials in humans. It therefore follows that a drug development project must be undertaken with due consideration to appropriate regulatory guidelines. These guidance documents are not intended to establish legally enforceable requirements, but rather reflect the current thinking of the respective agencies. The most scientifically sound and ethically correct approach for the particular “Target Product Profile” (TPP) under development should be adopted. Agent-directed, preclinical studies should be designed so as to support the conduct of clinical studies that may follow [81]. Pre-clinical modelling is aimed a sparing time and resources. The principle outcome is to treat humans. Assumptions are made that pre-clinical data is predictive of activity in humans – therapeutic index (efficacy vs. toxicity).
The value of any model is thus based on its ability to be predictive of clinical responses. An internationally-harmonised document, ICH S9 [82], was recently released (29 October 2009) for final review (Step 4) by the respective regional agencies: USA – Food and Drug Administration (FDA); Europe – European Agency for the Evaluation of Medicinal Products (EMEA); Japan – Ministry of Health, Labour and Welfare (MHLW) before adoption. It is important to realize that ICH S9 is intended to enhance and provide clarity [83] to the earlier guidelines implying DeGeorge et al. [84]; CPMP/SWP/997/96 [85] and Nakae et al. [86] representing the recommendations of the FDA, EMEA and MHWL respectively. These documents are unique in that most guidelines, including those of the local (South African) regulatory authority – the Medicines Control Council (MCC), either explicitly or implicitly exclude cancer therapies from their recommendations.

TABLE OF CONTENTS :

• DECLARATION BY CANDIDATE
• ACKNOWLEDGEMENTS
• SUMMARY
• TABLE OF CONTENTS
• GLOSSARY OF ABBREVIATIONS
• 1. Introduction
• 2. Literature study
  • 2.1. Multidrug resistant (MDR) cancer and circumvention thereof
  • 2.2. Combination chemotherapy
  • 2.3. Riminophenazines
  • 2.3.1. Background
  • 2.3.2. Clofazimine: Antineoplastic and Chemosensitizing potential
  • 2.3.3. Tetramethylpiperidine (TMP)-substituted Riminophenazines
  • 2.3.4. Pharmacodynamics: Anticancer multi-mechanism
  • 2.4. Refinements in the pharmacokinetic usage of anticancer drugs
  • 2.4.1. Loco-regional chemotherapy
  • 2.4.2. The Enhanced Permeability and Retention (EPR) effect
  • 2.4.3. Lipiodol Ultra-Fluid
  • 2.4.4. Intelligent formulations: Nanoparticulate drug delivery systems
  • 2.5. Pre-clinical development of anticancer drug products: Regulatory perspectives
• 3. Target product profile, critical path and pre-clinical development plan
• 4. Riminophenazine procurement, purification and authentication
  • 4.1. Materials
  • 4.2. Authentication of Riminophenazine purity and integrity
• 5. In vitro antiproliferative bioassays
  • 5.1. Introduction
  • 5.2. Materials
  • 5.3. Methodology
  • 5.3.1. Cell culture preparation and in vitro antiproliferative assays
  • 5.3.2. Fixed molar ratio drug combination studies
  • 5.3.3. Assessment of additional ABC transporter inhibition
  • 5.3.4. Neoplastic specificity
  • 5.3.5. Ethical considerations
  • 5.4. Results
  • 5.4.1. COLO 320DM neoplastic cell cultures
  • 5.4.2. HCT-15 neoplastic cell cultures
  • 5.4.3. ASH-3 cell line
  • 5.5. Discussion and conclusion
• 6. Development of novel Nanoparticulate Drug Delivery Systems (NDDS)
  • 6.1. Introduction
  • 6.2. Materials
  • 6.3. Methodology
  • 6.3.1. Development of Riminocelles™
  • 6.3.2. Development of RiminoPLUS™ Imaging
  • 6.4. Results
  • 6.4.1. Riminocelles
  • 6.4.2. RiminoPLUS Imaging
  • 6.5. Discussion and conclusion
• 7. In vivo models of experimental toxicity and oncology
  • 7.1. Introduction
  • 7.2. Materials
  • 7.3. Methodology
  • 7.3.1. Pilot safety and acute in vivo toxicokinetic assessment of Riminocelles
  • 7.3.2. Toxicity marker profiling
  • 7.3.3. Efficacy assessment
  • 7.3.4. Ethical considerations and committee approval
  • 7.3.5. Statistical analysis
  • 7.4. Results
  • 7.4.1. Acute toxicokinetics
  • 7.4.2. GLP repeat dose toxicity
  • 7.4.3. Efficacy assessment
  • 7.5. Discussion and conclusion
• 8. Development and application of an optimised and validated LC-MS/MS method
  • 8.1. Introduction
  • 8.2. Materials
  • 8.3. Methodology
  • 8.3.1. Stock solutions, calibration standards, quality control and recovery samples
  • 8.3.2. Sample preparation
  • 8.3.3. Chromatographic conditions
  • 8.3.4. Mass spectrometric conditions
  • 8.3.5. Validation procedures
  • 8.3.6. Application in pharmacokinetic study
  • 8.4. Results and discussion
  • 8.4.1. Chromatography and Mass spectrometry
  • 8.4.2. Assay validation parameters
  • 8.4.3. Method application
  • 8.5. Conclusion
• 9. Final discussion and conclusions
• References
  • Appendix A. AUCC approval letters
  • Appendix B. Review of Riminophenazine QSAR
  • Appendix C. Pharmacokinetic and tissue distribution study schedule

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STRATEGIC PRE-CLINICAL DEVELOPMENT OF RIMINOPHENAZINES AS RESISTANCE CIRCUMVENTING ANTICANCER AGENTS