Tuesday, July 20, 2010

IN THE LAB - In Vitro Assays | Alternative to Animal Testing Saves Time, Money: In vitro irritection assay system can reduce such testing

By Brian Dell
Alternative to Animal Testing Saves Time, Money

The drive to refine, reduce and/or find alternatives for animal-based testing of pharmaceuticals, cosmetics, and personal care products has significantly accelerated the demand for validated in vitro studies that are viable equivalents to animal testing. The move to in vitro testing formats is hastened further by the high development costs and lack of assay robustness typical of animal-based testing, such as the long-established Draize test, which evaluates the acute toxicity of pharmaceutical or cosmetic products. By using in vitro assays in lieu of the traditional and often controversial practice of animal testing, manufacturers are realizing benefits characterized by the four Rs: reduction, refinement, replacement, and reproducibility.

With the alignment of certain factors—cost reduction, performance optimization, and a changing culture of acceptability around animal testing—in vitro assay formats will continue to gain acceptance by both product developers and the regulating bodies that ensure product safety.
A Time for Change

Over the last century, in vivo testing emerged as the preferred method to evaluate toxicity and to provide information on the potential dangers to humans from chemicals, pharmaceutical products, and raw materials. Until recently, animal testing was a common “gate” through which most products had to pass before being cleared for human use. Several factors have evolved the scientific community’s thinking and approach to animal testing, however.

First, ever-mounting pressure on manufacturers to cut costs and timelines to market is forcing researchers to find more efficient assays. The costs involved in caring for laboratory animals and developing and conducting validated animal studies are substantial, limiting the ability of young companies to compete. For example, with drug development, the timeline to develop and complete current good manufacturing practices-compliant preclinical studies can add years to a drug’s launch date. Thus, current market forces reward those manufacturers who find effective ways to do more at reduced cost and without sacrificing consumer safety.

The second factor driving the increased use of in vitro test methods is the availability of new assay and screening technologies, which offer substantial benefits over in vivo platforms. With the technical advances of the last 30 years, in vitro test methods demonstrate superior efficacy as defined by the four Rs.
RabbitWith the alignment of certain factors—cost reduction, performance optimization, and a changing culture of acceptability around animal testing—in vitro assay formats will continue to gain greater acceptance by both product developers and the regulating bodies that ensure product safety.
Goal of Today’s Researchers

In vitro testing offers a compelling business case. Improved reproducibility and consistency in lot-to-lot test data yield highly correlated results. The opportunity to administer assays in a high- throughput format means a quicker turnaround time for results—typically 48 hours vs. two to three weeks with traditional in vivo methods. These efficiencies sufficiently reduce costs, which enables testing of smaller amounts of more substances at an earlier development stage.

The technical advances afforded by modern in vitro test methods make it feasible to reduce the number of animals used for testing, refine the historical data provided by in vivo testing methods, replace existing in vivo tests with cheaper, faster in vitro approaches, and ultimately deliver a reproducibility level that is unobtainable with animal-based test methods. Several in vitro assays have been proven to achieve these goals.

A prime example of a proven in vitro alternative is the use of the Irritection Assay System (Celsis International Ltd.) as a substitute for the Draize test. Since the 1940s, the Draize test has used ocular and dermal testing on live rabbits to gauge acute pharmaceutical product toxicity. The Irritection Assay System is a standardized and quantifiable alternative model that detects and predicts the ocular or dermal irritation of potential raw materials or compounds.

For new pharmaceuticals, the Irritection Assay System evaluates the ocular and/or dermal irritation caused by a test sample. During the testing process, a proprietary protein solution is placed in the well of a Petri dish to mimic the eye proteins, and a membrane disk is placed on top of the protein layer. Different volumes or dilutions of test sample are incubated on the membrane; the membrane is observed for deterioration, and the remaining material in each well is mixed and tested for the degree of turbidity against a standard curve through optical density. After density scores are compared, the irritancy score is calculated.

Early in product development, an in vitro assay such as the Irritection format can determine whether animal testing is necessary. When Irritection results are positive for irritation or corrosion, there is no need to prove this again in an animal model. The pharmaceutical can be discarded, or the manufacturer can reformulate and reevaluate it using the Irritection test format. This practice reduces the total number of animals used in the research phase of product development. Additionally, both time and money are saved by the early determination of a product’s irritancy score. Working toward the same decision point in animal studies is significantly more time-consuming and expensive.
Refining in Vitro Methods

In vitro assays also serve to refine in vivo test information, which typically suffers from high variability across animal subjects. A prime example of an in vitro assay format that plays a refining role is the bacterial endotoxins test (BET: USP <85>). BET was originally designed to replace the rabbit pyrogen test for detecting Gram-negative bacteria in a test sample. Gram-negative bacteria are pyrogenic and raise body temperature by inducing the inflammatory response. Consequently, manufacturers must prove that pharmaceutical products intended for parenteral administration are clear of pyrogenic contamination prior to clinical use.

The U.S., European, and Japanese Pharmacopeias currently recognize two test methods for pyrogen testing. The rabbit pyrogen test (USP <28>) involves measuring the rise in temperature in rabbits following intravenous injection of a test solution. The febrile responses of rabbits to intravenous pyrogen vary from rabbit to rabbit due to the individual sensitivities of different animals. It is impossible to test certain drugs like cytokines, antibiotics, select sedatives/analgesics, plasma proteins, and radiopharmaceuticals this way. Variability, applicability, and a lack of standardized controls limit the accuracy of rabbit testing.

The in vitro BET is based on the coagulation of Limulus amebocyte lysate following endotoxin exposure. Immune cells in the blood will recognize pyrogens and release measurable fever-inducing signal molecules. Standardized controls and lot consistency eliminate the variability risk posed by animal subjects. BET predicts human response to pyrogens based on human fever, a method that is more relevant and accurate than those using animal analogs. While BET is limited to detecting only endotoxin pyrogens, more people are recognizing its ability to produce refined test data. Although some products still require animal testing, BET is now mandatory for many USP monographs.
RabbitIn vitro test methods ... are, consequently, gaining greater acceptance as strategic and necessary tools to reduce, refine, and replace animal use in drug research and product testing.
Replacing Animals with In Vitro Assays

Replacement occurs when an in vitro assay is deemed acceptable as an equivalent alternative to the animal test. The FDA has advocated for in vitro testing during drug development to assess safety issues like toxicity and drug-drug interactions. The FDA Guidance to Industry regarding drug interaction studies outlines the use of cellular and subcellular products for determining the inhibition and induction of key metabolizing enzymes. 1

Products such as cryopreserved hepatocytes or microsomes from human donors fulfill the requirements for pharmaceutical companies to assess these safety concerns. In this way, pharmaceutical companies can reduce the new drug attrition rate in clinical studies for safety and effectiveness issues by screening new chemical entities early in the discovery stage, then testing drug compounds prior to exposure in humans and animals, thus saving the company time and money while reducing the potential for adverse side effects in patients.

A final but also significant benefit of in vitro testing is the high lot-to-lot reproducibility and consistency of its test results. Many examples of variability have been noted when animals are used in testing. In fact, the biggest criticism of the Draize test is that it is unreliable and imprecise—the range of dermal responses among test animals extends from no effect to a very drastic skin irritation that includes necrosis. Studies suggest that the Draize test is only effective as a crude tool to distinguish irritants from non-irritants. The introduction of validated in vitro models such as Irritection and BET allows more consistent, measurable, and reproducible testing.

In conclusion, in vitro test methods deliver enhanced reproducibility and consistency in comparison to animal testing and are, consequently, gaining greater acceptance as strategic and necessary tools to reduce, refine, and replace animal use in drug research and product testing. As global manufacturers strive to deliver regulatory-compliant products to market in ever shorter time frames, in vitro methods such as the use of cryopreserved cellular research products and the Irritection and BET assays are not only viable alternatives to animal testing, but they are also strategic tools that can significantly reduce product development timelines and enhance the economic outcomes of the companies who use them.

Dell is associate director, biology, at Celsis Analytical Services. Reach him at bdell@celsis.com or (314) 885-1163.

1. United States Food and Drug Administration. Guidance for Industry: Drug Interaction Studies Study Design, Data Analysis, and Implications for Dosing and Labeling (2006 Draft). FDA. Available at: www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf. Accessed June 13, 2010.

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