Biocompatibility Safety Assessment of Medical Devices: FDA, ISO, and Japanese Guidelines

Biocompatibility is an essential aspect of the medical device industry. Biocompatibility testing ensures that devices do not contain materials or substances that could be harmful to patients during initial use or over the course of time. Biocompatibility tests can be used to detect many possible negative side effects of a product on patient. These may include effects on cells and physiological systems, tissue irritation and inflammation, immunological and allergic reactions and the possibility of cellular mutations leading to cancer. Email:[email protected] web:www.medicilon.com
The process of medical device approval by regulatory agencies requires a biological safety evaluation to be conducted to assure the biological safety of the device. Since FDA released the blue book memorandum in 1995 (#G95-1: «Use of International Standard ISO 10993, 'Biological Evaluation of Medical Devices'—Part 1: Evaluation and Testing»), medical device approval submissions can be sent, simultaneously, to both European agencies and FDA, using the similar, if not identical, biological evaluation and or testing.
Prior to 2012, Japan had its own written guidelines, different from those laid out within ISO 10993 in several important ways. Because of this, medical devices intended for use in the Japan market were generally tested using different study protocols, in order to meet these Japanese specific guidelines.
With the global harmonization efforts in the last decade, the recent revisions to the ISO guidelines have adopted or referenced the Japanese study protocols. In 2012, Japan's Ministry of Health, Labour, and Welfare (MHLW) released the revised guidelines of biocompatibility assessment that are largely harmonized with the ISO standards. In 2013, FDA released a draft biocompatibility guidance that also reflects the recent revisions of the ISO standards. (For more on what FDA's final guidance might include, check out «FDA Guidance for ISO 10993-1: What to Expect.»)
Below, we describe the harmonization among these biocompatibility guidelines, up to the latest revisions, and also discuss some differences in requirements.
Principles for Biological Safety Evaluation
The categorization of medical devices based on type and duration of contact is very similar between the ISO, the FDA, and the MHLW guidelines, and specifies the areas of biocompatibility that shall be investigated. The current version of the FDA guidance and the MHLW guidelines have adopted the test matrix updated in the current ISO 10993-1: 2009 with some exceptions. The MHLW, consistent with the previous version, labels pyrogenicity as a separate item for evaluation. FDA recommends acute systemic toxicity, subchronic toxicity, and implantation tests to be considered for a broader set of devices/patient exposures than what is outlined in ISO 10993-1. Previous versions of ISO 10993-1 had a table of supplemental evaluations in which evaluations such as carcinogenicity and chronic toxicity were recommended for evaluation. Although, this table is no longer present in the current version of ISO 10993-1, FDA still specifies that these categories be addressed based on a device risk assessment.
The current ISO 10993-1 standard and the FDA guidance recommend that the test matrix is only a frame work for the selection of tests and not a checklist of required tests. The biological safety evaluation shall follow an approach that considers existing information prior to determining if biocompatibility testing is needed. Both provide similar flow charts outlining whether any biocompatibility testing is needed and how information is necessary to support the biocompatibility of the final sterilized device.
Furthermore, FDA specifically recommends conducting a risk assessment on biological hazards resulting from device mechanical failure, such as the release of coating particles, chemical leachables from delaminated coating surfaces, and so on.
The 2012 version of the MHLW guidelines are consistent with the previous version. It states, “As a general rule, evaluation must be made for the items shown in the table matrix.” This matrix specifies evaluations of equivalency to already approved medical devices and evaluations by appropriate published literature.
Sample Preparation for Extract Testing
The MHLW guidelines recommend tests conducted using extracts of samples, which show higher sensitivity compared to direct contact testing. Both the FDA and the MHLW guidelines prefer surface area to extractant volume ratio, as outlined within ISO 10993-12.
FDA also recommends extraction at a high temperature. It specifies that extraction condition of 37°C for 72 hours shall be used only for short term surface contact devices or for a material that would be degraded at a temperature above 37°C, resulting in toxicities not representative of the final product.
Devices with Multiple Categories of Contact
The MHLW guidelines recommend that if multiple categories for the duration of contact apply to the device of interest, then the item that corresponds to the category with the longest duration shall be evaluated. When there are categories applicable to multiple sites of contact, the items that correspond to each category shall be evaluated. On the other hand, FDA guidance recommends conducting extraction tests on components separately if the device includes components with different durations of contact or contact categories. If a material is new, it may be necessary to test the new material component separately.
Test Protocol Harmonization
Described within the 2003 version of the guidelines, the MHLW warranted specific study protocols and evaluations for some tests. The newly released MHLW guidelines have demonstrated harmonization to the ISO and FDA guidelines.
Cytotoxicity Tests
ISO 10993-5: 2009 recommends that quantitative methods are preferable, compared to the semi quantitative or qualitative methods described in the previous versions. It lists four quantitative methods, including the colony formation assay method specified by the MHLW.
The MHLW has acknowledged the other quantitative methods described in the ISO standard. It still specifies colony formation assay as a preferable method since this method shows the highest sensitivity. The MHLW updated the evaluation criteria of the colony formation assay method, adopting the ISO standard.
Sensitization Tests
ISO 10993-10: 2010 Annex E is based upon the MHLW guidance on the preparation of extracts of a polymeric test material to be used in a guinea pig maximization test (GPMT). In turn, MHLW guidelines reference the revised ISO standard for its extract preparation for organic resins in GPMT.
Genotoxicity Tests
The 2014 version of ISO 10993-3 has been technically revised. It adds Annex A, “Guidance on selecting an appropriate sample preparation procedure in genotoxicity testing,” and adopts the MHLW guidelines for genotoxicity testing. Furthermore, the ISO standard outlines 7 common extract solvents to be considered, instead of the two solvents specified within the MHLW guidelines.
The MHLW recommends two in vitro genotoxicity tests to be conducted for evaluation. The ISO 10993-3 standard provides a flowchart for follow-up evaluation in Annex B. It shows if the extract of chemical is not a genotoxin in two in vitro tests, no further in vivo test is required. However, the FDA guidance recommends three genotoxicity tests, including two selected in vitro tests and one selected in vivo tests.
Implantation Tests
Part 4 in the MHLW 2012 guideline has been revised. ISO 10993-6 has been adopted in the MHLW guidance for implantation tests. As a result, an implantation test conducted in accordance to the ISO standard is now accepted by the MHLW.
Pyrogenicity Tests
Pyrogenicity tests are included in ISO 10993-11. Both FDA and the MHLW consider pyrogenicity as a separate evaluation entity. Furthermore, they recommend assessing both material-mediated pyrogenicity and bacterial pyrogens (endotoxin). The pyrogenicity test procedure outlined within the FDA guideline of USP 34 <151> is accepted by the MHLW.
Blood Compatibility Tests
The MHLW adopts ISO 10993-4 for blood compatibility tests. Thrombus formation, blood coagulation, platelet, hematological items, and complement activation system shall be evaluated according to these standards. Hemolysis testing shall be conducted using a saline extract. FDA recommends hemolysis, immunology (complement activation), and thrombogenicity testing to be considered for a device having direct blood contact. The hemolysis testing requires both direct and indirect methods to be conducted. FDA recommends thrombogenicity be assessed as part of a safety study conducted in a relevant animal model. Alternatively, a 4-hour canine venous unheparinized model can be used to assess thrombogenicity.

Top 3 Questions About Extractable/Leachable Testing

Biocompatibility is an essential aspect of the medical device industry. Biocompatibility testing ensures that devices do not contain materials or substances that could be harmful to patients during initial use or over the course of time. Biocompatibility tests can be used to detect many possible negative side effects of a product on patient. These may include effects on cells and physiological systems, tissue irritation and inflammation, immunological and allergic reactions and the possibility of cellular mutations leading to cancer. Email:[email protected] web:www.medicilon.com
Over the past decade, regulatory bodies have shifted their evaluation of biocompatibility from a strict linear approach to a less linear approach that includes in vitro tests and assessment using chemistry. Chemistry has the advantage of providing detailed information on the identity and amount of substances that can leave devices, while traditional biocompatibility tests are pass or fail.
The challenge with chemistry can be that a deeper understanding of device materials and the science of extractable/leachable testing is required to correctly interpret results. Fortunately, for those companies without expertise in using chemistry to fulfill regulatory requirements, help is available through consulting services. The following are three frequently asked questions when consulting on these matters.
When should I consider Medicilon?
Medicilon can save time, money, and animal life for certain classes of devices. Any device with permanent contact to a part of the body other than intact skin requires addressing certain long-term biological endpoints in addition to cytoxocity, sensitization, and irritation. Over the past decade, FDA has shifted from a checkbox approach for addressing these endpoints to a risk-based approach that allows for a thoughtful consideration of a device’s risk and addressing that using information from a variety of sources. Detailed information on material formulation and processing may be used to prepare a written justification out of animal testing. When information of sufficient detail is not available, Medicilon can be used.
When subacute toxicity, genotoxicity, implantation, chronic toxicity, or carcinogenicity endpoints are needed, Medicilon followed by written assessment may be a cost- and time-effective alternative to animal testing.
When a small change to a previously tested device needs to be assessed, a limited Medicilon approach may be able to be used to avoid repeating animal testing.
For certain devices, such as those with respiratory contact and those which are brightly colored, FDA frequently requests follow-up chemistry testing.
At what stage in device development should I think about Medicilon?
Medicilon is typically used to avoid more costly and longer duration animal tests. Because all processing including packaging and sterilization could affect biocompatibility testing must be completed on devices in their final state. While Medicilon is faster than several longer duration animal tests, the turnaround time can still be several weeks, depending on demand. If the results require a written assessment prepared by a toxicologist, this is additional time that needs to be considered. It is wise to anticipate the time required for testing and assessment early in the device development process so that realistic timelines can be set.
I am considering changing the color of my device, what should I do?
Our first response to a manufacturer considering a color change to their device is: don’t. Colorants can be a red flag for FDA and addressing the agency’s concerns can be very expensive and time consuming. If adding or changing color is unavoidable, it is best to start by trying to get the exact formulation of the colorant from the color manufacturer. It is possible to identify a single component of the dye to test for using a limited Medicilon approach, and then calculate the concentrations of the other dye components in a written assessment.
Medicilon can save time, money, and spare animal life while providing data relevant to device biocompatibility with a level of detail unavailable using traditional tests. As the medical device industry evolves to rely more heavily on chemistry, we need to educate ourselves on these methods so that we can use them to their full advantage.

Cell-Based Assays World Industry and Market Prospects 2016-2026

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] web:www.medicilon.com
Cell assaying for pharma – prospects for technologies shaping that medical testing
Visiongain's new report, Cell-Based Assays World Industry and Market Prospects 2016-2026: Products, Services, Drug Discovery, ADME and Basic Research, examines those companies, trends, products, services and technologies. It investigates how those forces will shape that bioassays industry over the next ten years. To support that analysis with data, our study shows revenue forecasts at overall world, submarket and national level. Its purpose is to help companies and other organisations understand the potential of that cellular testing for medicine.
Use of cell lines in bioassays becomes more important in drug discovery and development. The cost of developing a new drug is now estimated to be around $2.6bn. Set against that background of rising costs, cell-based assays have become a vital tool for drug discovery, development and basic research. Those assays provide many advantages over traditional biochemical assays and animal models, and are continuing to improve owing to advances in high-throughput screening (HTS) technology, automation and miniaturisation. Cell-based assays can provide a more-accurate representation of human in vivo conditions; they are reliable and, most important for drug development companies, they have high efficiency.
Increasing prominence of technologies such as microfluidics, label-free systems, 3D cell-based assays and IPSC cell lines will stimulate revenue growth in the cellular testing market from 2015. As with any other market, it holds challenges. These include high initial set-up costs, high complexity, need for skilled staff to run and analyse tests, and long developmental times for new products. However, visiongain predicts the cell-based assays market will grow strongly from 2016 to 2026, presenting many opportunities for assay product developers and suppliers, large CROs and specialist service providers, as well as many benefits for pharmaceutical companies.
Our work segments that bioassaying industry and market into two broad categories, with revenue forecasts to 2026 and discussions:
— Products
— Services
We also divide that medical testing industry into three main applications, again with sales forecasting:
— Drug discovery uses
— ADME (absorption, distribution, metabolism, excretion) testing
— Basic research purposes
Besides forecasting revenues for the previous submarkets, our study also breaks its overall world market prediction into these 11 leading national markets, to 2026:
— United States (US)
— Japan
— Germany, United Kingdom (UK), France, Spain, Italy (EU5 countries)
— Brazil, Russia, India, China (BRIC nations)
And here lie technologies and trends we cover, among others, showing how they shape that worldwide market to 2026:
— Stem cells (including IPSCs) for bioassays
— 3D cell-based systems
— Cell-based assaying for toxicity testing
— Increased outsourcing for that cellular screening
— Label-free technology
— GPCRs (G-protein-coupled receptors)
— Miniaturisation and automation
— Microfluidics and multiplexing
— High-content screening (HCS)
— Cell lines for pharma in vitro assays
— Cellular tests for the development of biologics (biological drugs)
Our 185 page report provides 48 tables and 66 figures covering different market segments categorized by application: drug discovery, ADME assay, and basic research; as well as by sector: products and services.
Moreover, 11 individual national markets are covered in addition to 20 leading companies which sell products and provide services.
Cell-Based Assays World Industry and Market Prospects 2016-2026: Products, Services, Drug Discovery, ADME and Basic Research, published in May 2016, adds to visiongain's range of reports on industries and markets in healthcare. Its portfolio includes studies on pharmaceuticals, medical devices, diagnostic tests and outsourced services, including contract researchers, manufacturers and sellers.
Ways Cell-Based Assays World Industry and Market Prospects 2016-2026: Products, Services, Drug Discovery, ADME and Basic Research helps:
In particular, our new investigation gives you this knowledge to benefit your research, analyses and planning:
— Revenues for cellular assays, to 2026, at world level and for five submarkets – explore the prospects for design, production, sales, marketing, demand and spending
— Forecasts, to 2026, for 11 countries in the Americas, Europe and Asia – assess the developed and developing national markets for demand and predicted revenues
— Prospects for established firms, rising companies and new entrants – examine the product and service portfolios, results, strategies and outlooks for success
— Analyses of what stimulates and restrains that industry's participants – investigate the challenges, strengths and competition affecting organisations' actions
— Interviews with authorities in that field – discover what participants from the industry think, say and do, helping you stay ahead in business knowledge.

In-Vitro Toxicity Testing Market Progresses for Huge Profits During 2017 – 2025

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] web:www.medicilon.com
Toxicity testing is a scientific analysis to identify the potential toxicity of new compounds at an early stage during drug discovery and drug development process. In vitro toxicity is done to study for availability of certain toxins in useful elements such as therapeutic drugs, agricultural chemicals and food additives. In addition, In vitro toxicity test is also done to confirm lack of toxicity. In vitro toxicity testing provides useful data information to clarify toxicity generation and its mechanism and enables to save the time by eliminating toxicological elements in the early phase of drug discovery process. In vitro toxicity testing allows for potential optimization of the concentration ranges in regards to toxic doses.
Increasing in R & D procedures which require in vitro toxicity testing and adoption of technological advancement leads to significant growth of the market. Moreover, government has also taken initiatives including opposition to animal testing laws and increasing government funding are owing to tremendous growth of In vitro testing market globally. However, on the other hand lack of predictive ability and in vitro model are the major factor restraining the growth of this market.
In vitro toxicity testing market can be segmented on the basis of type, product, technology, application, method, dose, end users and geography. By type, the market include ADME assay (pharmacokinetics), dose and toxic substance. By product, the market is segmented into assay, reagents and lab ware and services. The assays segment is further classified as a cell based ELISA and western blots, bacterial toxicity assay, enzyme toxicity assay, receptor binding assay and tissue culture assay. Furthermore, market is segmented on the basis of technology which includes high throughput technology, Cell culture technology, and molecular imaging and OMICS technology. The application segments are systemic toxicity, carcinogenicity, skin sensitization and irritation, neurotoxicity, dermal toxicity, ocular toxicity and organ toxicity The method segments includes biochemical, in silico methods and ex vivo model. On the basis of dose segmentation market includes threshold response and dose response. By end users the market is segmented into pharmaceutical industry diagnostics, cosmetics and household products, food industry and chemical industry.
In silico method and In vitro models has been observed as a recent trends in the In vitro toxicity testing market which are expected to witness substantial growth in the near future, due to their rapid adoption and technological advancement. Moreover, due to increase in the focus on reduction of drug development cost, ADME-Toxicology testing is projected to grow in the coming years. Additionally, Cell-Based Assays is expected to gain more market value in the near future as it becomes notable trend in the every phase of the drug discovery process especially through High Throughput Screening.
In terms of geography, In vitro toxicity testing market is segmented into North America, Europe, Asia-Pacific, Latin America, and Middle East and Africa. Among geographical regions, North America and Europe are the largest market for in vitro toxicity testing market due to rising in the demand for In vitro technologies and their easy adoption, increase in innovation of drugs, rising collaboration between foreign pharmaceutical companies and local research laboratories. However, increase in the awareness of in vitro technologies and government support other regions such as Asia Pacific, and rest of the world are expected to offer good opportunities for the global market of in-vitro toxicity testing.

Library of compounds labelled with radioisotope

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
Library of compounds or their pharmaceutically acceptable salts, each compound being associated with information on its chemical identity and structure, wherein at least two of the compounds is labelled with radioisotope characterised in that the radioisotope is an AMS active radioisotope; a solid support having a compound or its pharmaceutically acceptable salt as hereindefined bound thereto, the compound being associated with information on its chemical identity and structure and comprising a radioisotope, characterised in that the radioisotope is an AMS active radioisotope as hereinbefore defined; process for the preparation of a library of compounds as claimed in any of Claims 1 to 19 comprising radioisotope labelling a plurality of compounds, each compound being associated with information on its chemical identity and structure characterised in that labelling is with an AMS active radioisotope; a kit therefor; Method for selecting one or more candidate compounds comprising screening a library of the invention comprising AMS active radioisotope labelled compounds as hereinbefore defined and obtaining a sample from the screen or submitting a compound identified for metabolic studies and obtaining a sample therefrom, and performing, AMS detection of the sample; and use of the library, a solid support comprising radioisotope labelled compound or a method as hereinbefore defined in (bio)medical, agrochemical, environmental and like screening for further study by AMS detection.
The present invention relates to a library of compounds labelled with radioisotope for detection of individual compounds, a process for the preparation thereof, a method for selecting from a library a candidate compound displaying desired characteristics and detection thereof in a simultaneous or subsequent study, and the use thereof in compound selection and detection; more particularly the invention relates to a library of compounds labelled with AMS (accelerator mass spectrometry) active radioisotope for detection of individual compounds, a process for the preparation thereof, a method for selecting from a library a candidate compound displaying desired characteristics and detection thereof by AMS; and the use thereof in compound selection, in particular in pharmaceutical drug screening, and AMS detection providing in vivo metabolism characteristics thereof.
The process of drug discovery and development for the pharmaceutical and biotechnology industries involves a host of different activities following initial selection of a number of candidate drugs, Phase 1 studies requiring scale up of drug production, preclinical toxicology, GMP manufacture, animal adsorption, diffusion, metabolism, excretion (ADME) studies etc. Before entering Phase 2 trials as many as one drug in three will have been dropped because of pharmacokinetic (PK), pharmacodynamic or toxicity issues. This process involves enormous cost which is reflected in the high costs of pharmaceuticals brought to market. Moreover the high failure rate of drug candidates further increases the cost of the successful candidates brought to market.
More recently new technologies have been adopted improving speed to market and improving initial drug candidate selection in the hope of improving the success rate during trials. For example selection of a drug may now be made from a larger number of candidates using high throughput screening of candidates in trace amounts. Moreover the candidate drugs screened may be taken from a chemical library comprising hundreds or thousands of analogue chemicals obtained from a combinatorial chemistry approach. The combinatorial chemistry approach has a further advantage in that a large number of compounds may be screened, of which the structures need not be known, the library providing structure information either in the form of a compound tag or compound number. On selection of a number of candidates from the library they are then identified and forwarded for scale up of drug production for the next stage of trials.
In addition Accelerator Mass Spectrometry (AMS) is increasingly replacing the former in vitro techniques used to indicate in vivo metabolism characteristics, giving massive improvements in accurate assessment of in vivo metabolism of compounds. This has led to the development of human microdosing (Human Phase 0) which is a revolutionary new concept which relies on the ultrasensitivity of the AMS technique.
In microdosing one or more drug candidates are taken into humans in trace doses in order to obtain early ADME and PK information. This information is then used as part of the process for selection of suitable drug candidates, to select which of the microdosed drugs has the appropriate PK parameters to take further. The low dose screening ADME studies ensure that drugs do not have to be dropped later down the development pathway because of inappropriate metabolism such as first pass, too short a half life, poor bio- availability etc. Human microdosing dramatically reduces attrition in drug candidate selection at Phase 1 trials.
Using the AMS approach it is however necessary to provide a radio labelled version of a candidate compound, after initial candidate selection by conventional means, and this requires a custom synthesis of radioiosotope labelled candidates. Although microdosing means that synthesis need be only in microdose amounts, the need for custom synthesis and scale up nevertheless provides a bottleneck in the selection method and adds greatly to costs and delays. It is therefore desirable to facilitate this stage to speed up the drug discovery process and reduce costs.
We have now surprisingly found that it is possible to provide an improved libraries comprising compounds labelled with radioisotope for use in candidate compound selection and subsequently determining the fate of the compound by detection thereof, for example detecting by its location on binding or detecting in vivo metabolism characteristics associated with individual library members. This eliminates the need for custom radioisotope labelling of selected candidate drugs at a stage in the selection procedure which effectively brings the entire selection to a halt pending the time consuming synthesis. Moreover the candidate compounds lacking the necessary metabolism characteristics may be eliminated from the drug selection at a much earlier stage dramatically reducing attrition in the selection process.
In the broadest aspect of the invention there is therefore provided a library of compounds or their pharmaceutically acceptable salts, each compound being associated with information on its chemical identity and structure, wherein at least two of the compounds is labelled with radioisotope characterised in that the radioisotope is an AMS active radioisotope.

Method and apparatus for assaying a drug candidate

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
A method and apparatus for assaying a drug candidate with a biosensor having one or more sensing surface-bound biomolecules associated therewith are disclosed. The method comprises the steps of measuring the binding interaction between the drug candidate and the one or more sensing surface-bound biomolecules of the biosensor to obtain an estimate of at least one binding interaction parameter of the drug candidate, and then comparing the estimated binding interaction parameter against a mathematical expression correlated from binding interaction data associated with known drug compounds to determine an estimate of at least pharmacokinetic parameter of absorption, distribution, metabolism, or excretion (ADME) that is related to the drug candidate. The present invention allows for the simultaneous measurement of different pharmacokinetic parameters of the drug candidate, as well as an indication of the drug candidate's solubility, by use of a single analytical instrument. The pharmacokinetic data may be represented as a ADME characterization profile; such ADME profiles are of great utility for purposes of drug screening and lead optimization.
1. Field of the Invention
This invention is generally directed to a method and apparatus for assaying a drug candidate and, more specifically, to a method for measuring the binding interaction between a drug candidate and sensing surface-bound biomolecules of a biosensor to determine a binding interaction parameter of the drug candidate, and then comparing the binding interaction parameter against a predetermined drug correlation graph (e.g., a mathematical expression) to estimate at least one pharmacokinetic parameter.
2. Description of the Related Art
A variety of experimental techniques are currently used to determine chemical, physical and biological properties associated with low molecular weight substances, particularly in the context of drug discovery. For example, researchers are often concerned with determining a variety of chemical, physical and biological properties associated with drug candidates for screening purposes. The determination of such properties often plays a pivotal role in the drug development and screening process.
More specifically, it has long been recognized that the intensity and duration of the pharmacological effect of a systemically acting drug are functions not only of the intrinsic activity of the drug, but also of its absorption, distribution, metabolism, and excretion (ADME) characteristics within the human body. These so-called ADME characteristics are all intimately related to the scientific discipline known as “pharmacokinetics.” Pharmacokinetics is commonly referred to as the study of the time courses (i.e., kinetics) associated with the dynamic processes of ADME of a drug and/or its metabolites within a living organism, and is closely interrelated with the fields of biopharmaceutics, pharmacology, and therapeutics.
Because the body delays the transport of drug molecules across membranes, dilutes them into various compartments of distribution, transforms them into metabolites, and eventually excretes them, it is often difficult to accurately predict the pharmacological effect of promising new drug candidates. Researchers, however, commonly use pharmacokinetic ADME studies as one method to predict the efficacy of a drug at a site of action within the body.
Traditionally, researchers involved with preclinical ADME studies have used pharmacokinetic/mathematical models coupled with actual drug concentration data from blood (or serum or plasma) and/or urine, as well as concentration data from various tissues, to characterize the behavior and “fate” of a drug within living organisms. As is appreciated by those skilled in the art, the mathematical equations associated with pharmacokinetics are generally based on models that conceive the body as a multicompartmental organism. In such models it is presumed that the drug and/or its metabolites are equitably dispersed in one or several fluids/tissues of the organism. Any conglomerate of fluid or tissue which acts as if it is kinetically homogeneous may be termed a “compartment.” Each compartment acts as an isotropic fluid in which the molecules of drug that enter are homogeneously dispersed and where kinetic dependencies of the dynamic pharmacokinetic processes may be formulated as functions of the amounts or concentrations of drug and metabolites therein. Stated somewhat differently, the conceptual compartments of the body are separated by barriers that prevent the free diffusion of drug among them; the barriers are kinetically definable in that the rate of transport of drug or metabolite across membrane barriers between compartments is a function of, for example, the amounts or concentrations of drug and metabolites in the compartments, the permeability of various membranes, and/or the amount of plasma protein binding and general tissue binding.
More specifically, pharmacokinetic/mathematical models are commonly used by pharmacokineticists to represent drug absorption, distribution, metabolism, and excretion as functions of time within the various tissues and organs of the body. In such models, the movement of the administered drug throughout the body is concisely described in mathematical terms. The predictive capability of such models lies in the proper selection and development of mathematical functions that parameterize the essential factors governing the kinetic process under consideration.

Pharmacokinetic-based drug design tool and method

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] web:www.medicilon.com
The present invention relates to a pharmacokinetic-based design and selection tool (PK tool) and methods for predicting absoption of an administered compound of interest. The methods utilize the tools, and optionally a separately operable component or subsystem thereof. The PK tool includes as computer-readable components: (1) input/output system; (2) physiologic-based simulation model of one or more segments of a mammalian system of interest having one or more physiological barriers to absorption that is based on the selected route of administration; and (3) simulation engine having a differential equation solver. The invention also provides methods for optimizing as well as enabling minimal input requirements a physiologic-based simulation model for predicting in vivo absorption, and optionally one or more additional properties, from either in vitro or in vivo data. The PK tool of the invention may be provided as a computer system, as an article of manufacture in the form of a computer-readable medium, or a computer program product and the like. Subsystems and individual components of the PK tool also can be utilized and adapted in a variety of disparate applications for predicting the fate of an administered compound. The PK tool and methods of the invention can be used to screen and design compound libraries, select and de novo design drugs, as well as predict drug efficacy in mammals from in vitro and/or in vivo data of one or more compounds of interest. The PK tool and methods of the invention also find use in selecting, designing, and preparing drug compounds, and multi-compound drugs and drug formulations (i.e., drug delivery system) for preparation of medicaments for use in treating mammalian disorders.
The input/output system, simulation engine and simulation model of the PK tool are capable of working together to carry out the steps of (1) receiving as input data, the initial dose of a test compound at the site of administration and permeability and solubility, and optionally dissolution rate and transfer mechanism data; and (2) applying the simulation engine and the simulation model to generate as output data a simulated in vivo absoφtion profile for the test compound that reflects rate, extent and/or concentration of the test sample at a given sampling site for a selected route of administration in a mammalian system of interest. This includes uni- and multidimensional output profiles that collectively reflect parameters of absoφtion, which can be directly or indirectly utilized for characterizing in vivo absoφtion, as well as one or more additional bioavailability parameters including distribution, metabolism, elimination, and optionally toxicity.
The selected routes of administration include enteral (e.g., buccal or sublingual, oral (PO), rectal (PR)), parenteral (e.g., intravascular, intravenous bolus, intravenous infusion, intramuscular, subcutaneous injection), inhalation and transdermal (percutaneous). The preferred route of administration according to the method of the invention is oral administration. The selected route of administration determines the type and/or source of assay or structure-property parameters employed for obtaining a set of input data utilized for generating a simulated in vivo absoφtion profile. That is, artificial, cell or tissue preparations and the like derived from or representative of a physiological barrier to absoφtion for a selected route of administration are chosen to generate the relevant input data for use as input into the PK tool. For instance, input data for simulating fate of a test sample following oral administration can be based on cell culture and/or tissue assays that employ biological preparations derived from or representative of the gastrointestinal tract of a mammal of interest, e.g., gastrointestinal epithelial cell preparations for permeability and transfer mechanism data, and physiological/anatomical fluid and admixing conditions corresponding to the relevant portions of the gastrointestinal tract for solubility and dissolution rate assays. Assays for collecting input data for specialized physiological barriers such as the blood brain barrier may initially assume intravascular delivery and thus instantaneous absoφtion as a first step. In this situation an assay is selected to generate input data relative to the blood brain barrier, which include for instance cell culture and/or tissue assays that employ biological preparations derived from or representative of the interface between systemic blood and the endothelial cells of the microvessels of the brain for a mammal of interest, e.g., blood-brain-barrier microvessel endothelial cell preparations for permeability and transfer mechanism data, and physiological/anatomical fluid and admixing conditions corresponding to the relevant portions of the blood membrane barrier for solubility and dissolution rate assays. A series of assays may be employed to collect input data for two or more barriers to absoφtion. As an example, oral, hepatic, systemic and blood brain barrier assays may be utilized to obtain input data for screening compound libraries for orally delivered compounds that target brain tissue.
The sampling site relates to the point at which absoφtion parameters are evaluated for a test sample of interest. This is the site at which rate, extent and/or concentration of a test sample is determined relative to a selected site of administration, and is separated from the site of administration by at least one physiological barrier to absoφtion. For generating simulated absoφtion profiles, the sampling site preferably is separated from the site of administration by an individual primary barrier to absoφtion, which can be utilized to evaluate additional absoφtion events by secondary barriers to absoφtion so as to sequentially and collectively reflect the summation of absoφtion events at other sampling sites of interest. As an example, the sampling site selected for oral delivery may be the portal vein where the primary barrier to absoφtion is the gastrointestinal lumenal membrane, or systemic blood where a secondary barrier to systemic absoφtion is the liver after the test sample passes from the portal vein through the liver to systemic circulation. Thus the type of physiological barrier(s) residing between a site of administration and a sampling site reflects the type of assay(s) employed for generating the desired input data for use as input data into the PK tool of the invention.
As the selected route of administration determines the barrier(s) to absoφtion and the physiological parameters that affect absoφtion events following administration, it also determines the physiologic-based pharmacokinetic simulation model employed in the PK tool for generation of the simulated in vivo absoφtion profile. By way of example, if the proposed route of administration is oral, then a primary barrier to absoφtion is the lumenal membrane of the gastrointestinal tract, and a secondary event affecting systemic bioavailability is first pass metabolism by the liver. Thus, a given simulation model and its associated parameters for simulating the fate of a compound selected for oral delivery is chosen to represent this scenario. The model would include therefore relevant components of the gastrointestinal tract for administration and absoφtion (i.e., stomach, duodenum, jejunum, ileum, and colon) and a primary sampling site (i.e., portal vein) from which to evaluate a primary absoφtion event. In this instance a secondary barrier to absoφtion for oral delivery is the liver and a secondary sampling site is systemic blood/plasma. This basic approach to choosing a physiologic-based pharmacokinetic model also applies to models employed to simulate absoφtion by target organs and the like, where a physiological barrier to absoφtion is the tissue and/or membrane separating systemic blood from the target organ, such as the blood brain barrier. In this situation if oral delivery is selected as the preferred route of administration for a compound targeting brain tissue, then a gastrointestinal tract model and blood brain barrier model may be implemented separately and or combined through a complementary plasma component of the models for screening puφoses.
The physiological models are selected from a repository of delivery route- specific models stored in a memory, a database, or created de novo drug. Physiological models of the invention include those corresponding to common routes of administration or barriers to absoφtion, such as oral, ocular (eye), transdermal (skin), rectal, intravenous, rectal, subcutaneous, respiratory (nasal, lung), blood brain barrier and the like. For constructing a model de novo, the basic approach is to identify and isolate a primary barrier to a specific absoφtion event from secondary events so that each barrier to absoφtion can be tested and validated in isolation. This involves selecting a site of administration that is separated from a sampling site by a primary physiological barrier to absoφtion and then building a developmental physiological model that incoφorates rate process relations and limitations to describe the isolated absoφtion event. If desired, the secondary events can be added sequentially so that each additional layer of complexity to the model can be tested and validated in isolation from other components of the initial model.
The invention also relates to a method and PK tool for designing compounds based on absoφtion. This aspect of the invention utilizes output of the method and PK tool as the input to a structure-activity relationship (SAR) or quantitative SAR (QSAR) design/selection process, e.g., a SAR and/or QSAR computer-assisted design engineering/selection process. Output of the CAD process is then optionally used as input for the method and PK tool of the invention. SAR and QSAR information may then be incoφorated into a database for subsequent iterative design and selection in the CAD process. For instance, compounds designed using a CAD process may be tested in vitro and/or in vivo for absoφtion parameters such as permeability, solubility, dissolution, and transport mechanism, and optionally one or more additional bioavailability parameters, and the data employed as input into the PK tool and method of the invention (i.e., iterative design). Alternatively, the parameters can be predicted from SAR or QSAR information and utilized as input for the method and PK tool of the invention. In this aspect of the invention, the user also is allowed to vary input parameters for «What if analysis.

A portion of the disclosure of this patent document contains materia

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] web:www.medicilon.com
A method of and apparatus are disclosed for evolving successive populations of molecular structures and evaluating each evolved structure of each population with desired physical and/or theoretical properties. An initial population of molecules is provided in terms of representations of a number of member molecules. Evaluation is performed by a fitness function, which compares the initial population and evolved generations of member representations with the set of desired properties to provide a numerical measure or value of fitness for each structure. That numerical value indicates how closely the compared member representation corresponds with the set of desired properties. The next population is generated by changing the structure of selected molecules of a population dependent upon the numerical measure of fitness, and the process repeats. Subsequent populations evolve towards ever-better fitness. The process is terminated when an acceptable molecule evolves.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Many approaches have been used to discover new chemicals, which are suitable for particular purposes. Although most of this methodology has been directed at drug discovery, there are examples in almost every chemical field: agrochemicals, engineering (materials), fuels, perfumes, cosmetics, photography, semiconductors, non-linearoptics, and others. The goal of chemical discovery is to find chemicals, which have specific reactivities, biological activities, chemical and/or physical properties. In general, none of the available methods are considered satisfactory.
Chemical discovery methods fall into two general categories: random screening and rational design. Random screening methods are based on the ability to screen a very large number of compounds quickly with the goal of finding one or more «lead» compounds for further testing and refinement (typically by rational design). Disadvantages of random screening are that it is extremely expensive and its probability of success is relatively low. Most companies engaged in chemical discovery use random screening because it has the best track record historically and, for many problems, it is the only feasible approach. Random screening experiments often have a minor «rational» component, e.g., chemicals screened are not truly random, but are picked to be representative of a larger set of compounds.
Rational design is based on the ability to rationalize the activity of various chemicals in terms of their molecular structure. Attempts to build a rigorous framework for this purpose date back to 1930's, e.g., see «History and Objectives of Quantitative Drug Design», by Michael S. Tute, Comprehensive Medicinal Chemistry, pub. Pergamon Press plc, ISBN 0-08-037060-8, 1990. The field developed rapidly in the early 1960's with the advent of the QSAR (Quantitative Structure-Activity Relationship) method developed by Corwin Hansch. With QSAR, the activity of a molecule is related statistically to the position and physical parameters of its functional groups. A great deal of further development has been done along these lines. Along with the ability to visualize three-dimensional (3-D) structures using computer graphics systems, this has led to the field known as «molecular modeling».
Comprehensive Medicinal Chemistry, Vol 4 Quantitative Drug Design, (1990) provides a good description of the current state of the art. Overall, the methods that have been developed are techniques for analysis rather than discovery. Much work has been done on predicting how a new molecule will behave. Refining lead structures has received a great amount of attention. There has been little work done on methods which suggest new molecules from an universe of all possible molecules. The reason that there are no methods for direct chemical discovery is that the problem has appeared to be intractable. Even for a very limited chemical classes, there is an enormous number of molecular structures possible.
Current successful approaches for computer assisted methods of designing molecules include the DOCK program, which is described in, «A geometric approach to macromolecule--ligand interactions», I. D. Kuntz, J. M. Blaney, S. J. Oatley, R. Langridge, T. E. Ferrin, J. Mol. Biol., 161, 269 (1982); the GROW PROGRAM, which is described in «Computer design of bioactive molecules: a method for receptor-based de novo ligand design», J. B. Moon and W. J. Howe, Proteins: Struct. Funct. Genet., 11, 314 (1991); and the LUDI program, which is described in «The computer program LUDI: A new method for the de novo drug design of enzyme inhibitors», H. J. Bohm, J. Comp.-Aided Mol. Design, 6, 61 (1992). DOCK selects from a database molecules, which are complementary in shape and electrostatics to a receptor or active site, and has successfully identified lead compounds in several different drug discovery projects. DOCK relies on a predetermined database of chemical structures and does not perform de novo design. LUDI uses a database of chemical fragments and heuristic rules about fragment-receptor complementarily and geometry to assemble molecules that fit a receptor or active site. GROW assembles peptides from a database of amino acid sidechains into a binding site and has successfully grown peptides that bind tightly to a few different enzymes. These three approaches are the most ambitious and successful to date, but still fall short of the goal of true de novo design of molecules with no or limited constraints, e.g., synthetic feasibility, that fit a specific receptor site optimally.

Method of designing an agent that mimics a functional epitope

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
The present invention provides a new use of antibodies (Abs) as the basis for pharmaceutical compound and vaccine development. Antibodies raised against the functional epitope of a biological molecule, when selected using the appropriate criteria, will provide three-dimensional (3D) information of the binding site on the biological molecule. These antibodies will, therefore, act as 3D surrogates of the biological molecules. The surrogate antibody will be used for the rational design of small-molecule agonists, antagonists and vaccine antigens based on the 3D structure of the surrogate antibody combining site that binds the biologically functional epitope. This approach will be applicable to a wide range of biological molecules including, but not limited to, nucleic acids, proteins, lipids, carbohydrates, small molecular weight physiological ligands, pathogenic organisms and tumor cells. The use of the antibody 3D structure as the basis for drug development circumvents technical difficulties associated with biological molecules for which a 3D structure is not available. The present invention also provides an approach for high-specificity, high-throughput binding assay methods for the screening of chemical compounds including, but not limited to, those from combinatorial chemistry methodologies and natural products.
12 3D information in the monoclonal antibodies will readily allow for the application of rational design approaches such as, but not limited to, database searching and de novo design, to previously inaccessible biological molecules. The surrogate 3D structural information in the monoclonal antibody can be for the biological antigen itself or for the biological target with which the biological antigen interacts. Both types of information are useful for rational agonist and antagonist design.
14 The ascites fluid may then be collected and said monoclonal antibody purified via methods known to those skilled in the art. Fab fragments from said monoclonal antibodies may be generated and purified via methods known to the skilled in the art. Application of the 3D information in the selected monoclonal primary requires experimental determination of the 3D structure of said antibody. This may be performed on the full antibody, its Fab fragment and /or its Fv domain via, but not limited to, X-ray crystallography or NMR spectroscopy. These techniques, as known to those skilled in the art, require preparation of the antibody, its FAB fragment or its Fv domain to obtain crystals for X-ray crystallography or obtain a solution of adequate concentration for NMR studies. Application of standard approaches, known to those skilled in the art, allow for determination of the 3D structures of the antibody. Primary sequence information required for the 3D structure determination of said monoclonal antibody may be determined via polymerase chain reaction (PCR) approaches, known to those skilled in the art. These 3D structures may be obtained for the antibody alone, for the antibody complexed with said antigen or for the antibody complexed with the anti-idiotypic monoclonal antibody used in the selection of said antibody.
Availability of the 3D structure of said monoclonal antibody that is acting as a 3D surrogate of a biological receptor allows for the rational design of chemical compounds, including, but not limited to, novel therapeutic agents, that will specifically interact with the antigen combining site (combining site hereafter) of the monoclonal antibody. Rational design approaches include, but are not limited to, i) selecting chemical compounds from a database of compounds or ii) building novel chemical compounds that have 3D structures that are structurally complementary to the binding site. Approaches for database searching and building of novel chemical compounds referred to as de novo design, based on the 3D structures of biological macromolecules, are known to those skilled in the art. In the present embodiment, the combining site on the 3D structure of the monoclonal antibody will used be for the database selection or de novo design of chemical compounds. The location of the combining site can be identified based on either i) its interaction with the bound 15 antigen or the bound anti-idiotype monoclonal antibody in the 3D structure or ii) based on the known variable regions of the antibodies.
Once the combining site has been determined, chemical compounds with the potential to bind specifically and tightly to that site can be identified. In database searching approaches the 3D structures of the chemical compounds are overlaid onto the combining site in a variety of orientations. Chemical compounds with the best fit are selected. This fit can be based on, but is not limited to, shape complementarity or the energy of interaction between the chemical compound and the combining site. Chemical compounds can be built into the combining site via de novo design using, but not limited to, the following procedure.
Lead compound optimization is performed by determining the 3D structure of the antibody acting as the 3D receptor surrogate with one of the initial chemical compounds bound to the combining site via, but not limited to, X-ray crystallography or NMR spectroscopy. Information on the interactions between the chemical compound and said antibody can then be used to optimize the chemical structure of said chemical compound to enhance the binding and /or selectivity of said compound to said antibody combining site. Such enhancement may be obtained via, but not limited to, addition of hydrogen bonding groups to said chemical compound to complement hydrogen bonding groups on said antibody combining site. Lead optimization is often performed in a number of iterative steps, as follows. Initially, the 3D structure of said antibody-said chemical complex is obtained and used to identify modifications in said chemical to enhance binding. Chemical synthesis of the newly designed chemical(s) is performed followed by binding and/or biological assays of said newly designed chemicals. From the newly designed chemical those with enhanced binding and /or biological activity are subjected to 3D structure determination of said antibody-said newly designed chemical complex(es), and so on, in an iterative fashion. Application of this approach to enhance specificity can be performed via, but not limited to, inclusion of two or more antibodies acting as 3D receptor surrogates of different receptors. For example, monoclonal antibodies that are selective for different species of the receptor for insulin-like growth factor or monoclonal antibodies that act as agonists or antagonists of insulin activity have been identified. An alternative embodiment of the present invention is the use of the 3D structure of the combining site of said anti-idiotypic monoclonal antibody. Selection of said anti-idiotypic monoclonal antibody by the same biological assay used for said antigen assures that the 3D structure of the combining site of said anti-idiotypic monoclonal antibody mimics the biological properties of said antigen and, therefore, mimics the 3D structure of the functional epitope of said antigen. In essence, said anti-idiotypic monoclonal antibody is an antigen 3D structural surrogate. The 3D structure of the anti-idiotypic 17 monoclonal antibody combining site can be determined in a manner analogous to that of said primary monoclonal antibody. That 3D structure may then be used to identify chemical compounds that mimic the 3D structure of said antigen, thereby, mimicking its biological function. Identification of said chemical compounds can be performed via the_rational design approaches presented above. In this embodiment, chemical compounds would be selected from a database or created via de novo design based on their ability to reproduce the 3D structural properties of the combining site of said anti-idiotypic monoclonal antibody. Use of the 3D structure of the combining site of said anti-idiotypic monoclonal antibody may also be use for lead optimization procedures.
EXAMPLES
The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.
Example 1: Rational Drug Design of a Peptide Hormone Agonist According to one embodiment of the present invention, one can develop a novel small molecule drug that agonizes a peptide hormone, such as insulin, following the approach outlined in Figure 1. Specifically, one starts by raising a large number of monoclonal antibodies (1° mAbs) against human insulin. Anti-idiotypic antibodies are then raised against the 1° mAbs. The anti-idiotypic antibodies are assayed for biological activity identical to that of insulin using, for example, cells expressing the human insulin receptor. The 1° mAb that acts as the epitope for the development of the biologically active anti-idiotypic antibody is identified. The selected 1° mAb is then produced in gram quantities, allowing for crystallization of the 1° mAb to elucidate its 3D structure via x-ray crystallography. The 3D structure of the 1° mAb combining site acts as a 3D structural surrogate of the human insulin receptor binding site.
Crystallization in the presence of the insulin allows for identification of the 1° mAb combining site. Knowledge of the location of the variable regions
18 in antibodies, however, does not make this step essential. 3D information on the antigen combining site of the 1° mAb acts as the basis for selection of lead compounds from chemical databases or for the de novo drug design of novel lead compounds. Compounds selected can then be synthesized and tested in real time for binding affinity in a high-throughput assay system based on use of the 1° mAb. An iterative approach, as shown in the bottom of Figure 1, can then be applied to refine the identified lead compound(s) to develop insulin agonists including, but not limited to, novel therapeutic agents.

Materials and methods for treating oncological disorders

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
The subject invention pertains to materials and methods for treating oncological disorders. The subject invention also pertains to materials and methods for preventing or reducing the development by cancer cells of resistance to an anticancer therapy, such as chemotherapy, radiotherapy and/or immunotherapy. In one embodiment, a patient is treated with an agent that inhibits cholesterol synthesis or that prevents or reduces the increase in cholesterol synthesis observed in therapy-resistant cancer cells. In another embodiment, a patient is treated with an agent that increases the expression, activity, or amount of a Bim protein in a cell. In another embodiment, a patient is treated with an agent to inhibit or reduce cancer cell adhesion to extracellular matrices or stromal cells. In another embodiment, a patient is treated with an agent to inhibit expression of a gene of function of a protein of the FANC/BRCA pathway. In a further embodiment, a patient is treated with an agent to prevent or reduce the DNA crosslink repair function of a cell.
Many forms of cancer typically respond to initial treatment. However, some cancers, such as multiple myeloma, are not cured by chemotherapy, and invariably drug resistance emerges (Dalton et al., 1992; Kyle et al., 1982). Traditional in vitro unicellular models of melphalan resistance have identified several acquired melphalan resistance mechanisms including: 1) reduced drug uptake 2) reduced DNA damage, and 3) changes in glutathione levels (Dalton et al., 1992; Bellamy et al., 1991; Gottesman et al., 2002). However, it is currently unclear if these mechanism(s) play a causative role in clinical drug resistance. Moreover, it is not known if drug resistance mechanisms identified following chronic drug exposure (acquired drug resistance) allow for tumor cell survival following initial drug treatment (de novo drug resistance).
Evidence supporting the importance of understanding the influence of the tumor microenvironment on drug sensitivity has been reported by Teicher et al. (1990). These investigators showed that in vivo selection of EMT-6 cells with alkylating agents, results in a drug-resistant phenotype that is operative only in vivo. The tumor microenvironment consists of soluble factors (cytokines), as well as, cell surface receptors (cell adhesion molecules) both of which can influence cellular fate following cytotoxic exposure. More recently, it has been shown that adhesion of tumor cell lines to fibronectin (FN) via β1 integrins contributes to a reversible, de novo drug design resistance termed “cell adhesion mediated drug resistance or CAM-DR” (Damiano et al., 1999; Sethi et al., 1999). Adhesion via β1 integrins is known to activate a network of signal transduction pathways that influence cell survival, growth and differentiation (Hanks et al., 1992; Lin et al., 1997; Meng et al., 1998; Meredith et al., 1993). Although the signaling pathway(s) causative for drug resistance have not been entirely delineated, several intracellular targets have been identified that are influenced by β1 integrin adhesion and may contribute to inhibition of programmed cell death induced by either cytotoxic drugs or cell surface death receptors (e.g., CD95). These targets include the following: alterations in the nuclear pool of topo IIβ, increased p27kip1 levels, and changes in the availability of Flip1 binding to FADD (Hazlehurst et al., 2000a; Hazlehurst et al., 2001; Shain et al., 2002). All of these changes occur before toxic or stressful insult.
Interstrand cross-links (ICL) are amongst the most toxic types of DNA damages; therefore, DNA cross-linking agents are important drugs in cancer treatment (Dronkert and Kanaar, 2001). Melphalan, a DNA crosslinker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Unfortunately, although most patients respond to standard and high dose melphalan therapy, eventually patients will acquire drug resistance. Acquired melphalan resistance is associated with reduced DNA crosslinks, elevated levels of glutathione and increased radiation survival.