Chronic (Repeated Dose) Toxicity
When dealing with chronic toxicity testing the aim is to assess the risk of therapy, establish a dose/effect relationship and to also establish safety margins if possible.
Again, this testing procedure uses two animal species, one of which is non-rodent. Usually a rat and a dog are used for testing. The dosage to be administered is carefully planned as to demonstrate toxicity. The duration of study is directly related to the proposed clinical use.
In general, one day of clinical use results in two weeks of animal study, and thirty days clinical use results in six months of animal study, and so on.
Usually three separate dose levels are used, where the animals are observed, sacrificed, and then undergo macroscopic and microscopic examination. In these cases some of the animals are usually kept alive to see if the effects of the drug are reversible.
Mutagenicity
Mutagenicity testing is used to determine the possible existence of mutagenic hazards. Any evidence of genetic damage raises concerns regarding carcinogenicity and cell damage. This method of testing is looking out for changes in the nucleotide sequence, chromosome (structures) and genone (number of chromosomes), changes. Mutagenicity usually comprises of four studies.
These are: The Ames test, which looks for mutation in salmonella. Testing for chromosome aberrations in human lymphocyte, Chinese hamster ovary cells. Ames testing is also carried out in mouse lymphoma and is used in testing rodent bone marrow for genetic damage. For all of the tests, the results require very careful and precise interpretation.
Carcinogenicity Studies
The objective is to estimate the carcinogenic hazard of use in man. This type of study is required if the drug is intended for chronic use (6 months), or frequent intermittent use. Study is also required if the chemical structure suggests carcinogenic potential. In this case the testing takes the form of “lifetime” studies in two species (rat for 2 years, and mouse for 18 months), at set dose levels which is usually the maximum tolerated dose.
Post mortem examinations are carried out, and histology of around 30 tissue samples are taken. The histology is also taken of any lesions on the animals who die on the study, as well as those who stay alive.
The results for this type of testing are very complex and are therefore usually difficult to interpret. Usually the best that can be done is to identify the potential for a drug to disturb regulatory mechanisms at high dosage levels in rodents.
Clinical Trials
If all of the desired specifications have been met up until this stage, the next procedure is clinical trials.
Phase I of the trials uses around 10 – 20 volunteers, where the drug is administered in single, repeated doses. This is to see how the human body handles the new drug. At this stage in the trials, testing is always placebo controlled.
Phase II involves the first patient trials and usually consists of testing on around 100 – 200 patients. The objective at this stage is to study efficacy and to obtain preliminary information on safety. Dose Ranging is also employed here, to establish optimum dosage. Some of the testing is placebo controlled at this stage.
Phase III involves thousands of patients and major efficacy and safety trials. Phase III is carried out under the same conditions as the marketed product, but under closer monitoring. This stage is actively controlled by placebo, and all results are compared with similar marketed treatments.
Phase IV is the final stage in the clinical trials. This stage takes place only after the licence has been granted. The drug is administered to different populations, such as the elderly and children. Post marketing surveillance is carried out to identify the occurrence of any rare adverse effects.
The final aspect to consider, before a license will be issued, does not involve sampling itself, but in fact deals with regulatory needs and clinical practice.
Good Clinical Practise (GCP) is an international ethical and scientific quality standard used specifically for the design, conduct, recording and reporting trials that involve humans.
Before any trial takes place, all foreseeable risks should weighed up against the anticipated benefits. Most importantly, the safety and well being of the subjects should prevail. All available non-clinical and clinical information on an investigational product should support trial. A clear and detailed protocol should exist, outlining information on every stage of the trials. In addition, trials should not commence without first being approved by a review board or ethics committee, whichever is appropriate.
Subjects who decide to take part in a trial must first give their full consent. The subjects must be given the right of total confidentiality and must only be treated medically by a fully qualified physician.
Good Laboratory Practise (GLP), is essential. GLP is a system of laboratory procedures, which are designed to ensure proper standards of work are being enforced. Also staff competence and record keeping are monitored in order to prevent error and fraud in the conduct of studies.
Good Manufacturing Practise (GMP) sets out a list of basic requirements that must be met. It states that all manufacturing processes are clearly defined, reviewed and shown to be capable of consistently manufacturing a product of the required quality and complying with specification. All instructions and procedures must be clearly defined.
Quality Assurance
Quality Assurance (QA) is a wide ranging concept which incorporates GMP. The system of QA was set up to ensure that a variety of factors are carried out.
QA ensures that all medicinal products are designed to comply with all the regulations set out by the GMP and GLP.
It ensures that arrangements are made for the manufacture, supply and use of the correct starting and packaging materials. Also, the QA checks that all necessary controls on intermediate products, and any other in-process controls and validations are carried out. The finished product must then be correctly processed and checked according to defined procedures.
The QA makes sure that medicinal products are not sold or supplied before a qualified person has certified release. It is further stated that a procedure for self-inspection must exist and that arrangements should be made to ensure that the products are stored, distributed and subsequently handled to ensue quality throughout shelf life.
Quality Control
Quality Control (QC) is part of GMP. There are a number of basic requirements that are set out by quality control, all of which should be adhered to.
It states that adequate facilities must exist, along with appropriately trained personnel and approved procedures for every part of the process. Retention samples should be taken and all test methods should be properly validated. Finally, records must be kept which demonstrate that all procedures were actually carried out, and the final product must comply with the marketing authorisation.
Regulatory Systems
There are a number of regulatory systems that must be consulted
before trials commence. The regulatory systems that are consulted vary, depending on the country in which the trials are being carried out.
In America, all medicinal products are regulated by the FDA (Food and Drugs Administration). This is done from the time that the drug is administered to the first human up until it is withdrawn from the marketplace. Before the first human can be dosed, an IND (Investigational New Drug Application) must be filed with the FDA for approval. The IND contains preclinical and manufacturing information and the protocol for the first trial. All preclinical and clinical study data should be presented at this stage. Once all the previous requirements have been met, a NDA (New Drug Application) can be applied for. This is a compilation of preclinical, manufacturing and
clinical information and should also include proposed labelling and pack inserts for the new drug.
In the UK, medicinal products are controlled by the MCA (Medicines Control Agency), from the first time they are given to patients until they are withdrawn from the marketplace. Before the first patient can be dosed a CTX (Clinical Trial Exemption), must first be filed with the MCA. A clinical trial exemption contains summaries of all preclinical, manufacturing and clinical data together with a clinical plan.
In the European Union there is a harmonisation of national legislation, as there has been an introduction of one set of legislative requirements. From 1995, the EMEA (European Medicines Evaluation Agency) has existed and a number of new procedures have been initiated. All of these regulations are governed by the use of directives for which the guidelines were produced.
Only after all of the afore mentioned procedures and regulations are met can a manufacturing and product licence be granted. For very obvious reasons of health and safety, this process must be strictly adhered to, and no exceptions can be made. The plight for obtaining licensing for a new drug is very rigorous and lengthy. The average time spent in research before a licence can be applied for is around 14 years.
Final Product Sterilisation
It is specified that the drug in question will be administered to patients via intramuscular injection. All finished product pharmaceuticals intended for parenteral administration and all surgical materials must be sterile (except for live bacterial vaccines). The presence of micro-organisms in final products is unacceptable for a variety of reasons.
The injection of a contaminated product would be likely to cause a severe infection in the recipient patient. This is also applicable to surgical materials. Micro-organisms may also be capable of metabolising the product itself, thus reducing its potency. This is particularly true of protein-based pharmaceuticals, as most of the microbes produce an array of extra cellular protinase enzymes.
A method of final product sterilisation is to be decided for our vaccine, and this should be done by selecting the sterilisation method that is most compatible with our drug.
Within the pharmaceutical and healthcare industries there are four main methods employed for final product sterilisation. These are Heat (Autoclaving), Ethylene Oxide gas, Gamma Irradiation and Sterile Filtration.
Heat (Autoclaving), guarantees sterility, but the product must be heat stable. This is a major drawback and makes this not a viable option for many biopharmaceutical products. This method is best suited to heat stable products such as saline solutions and surgical instrumentation.
Ethylene Oxide (EO) gas is used because the gas effectively kills bacterial spores and can kill viruses. Since EO is extremely penetrating it is the method of choice for solids, particularly plastics and implant materials that are not chemically modified by the gas.
The drawbacks for this method of sterilisation are that EO is highly reactive, extremely toxic and is very explosive in air. Also, following sterilisation it can be difficult to remove all traces of the gas, and can sometimes take up to 3 or 4 days to be removed.
With Gamma Irradiation, the sample is exposed to 2.5 Mrad of radiation from a source of 60C. This amount of radiation is generally recognised as a sterilising dose.
This method is highly effective, but the product must not be adversely affected by irradiation. Effect is that of free radical formation of species such as H+ and OH-, which are capable of inflicting biological damage. This method is most widely used for syringes, catheters, surgical gloves and sutures. Where possible gamma irradiation is
preferred to ethylene oxide because of the toxicity of the EO gas and the handling precautions.
Finally, we get on to sterile filtration. Sterile filtration is the method that is best suited for the sterilisation of our vaccine. This is because this method of sterilisation is excellent for solutions that are intended for injection. It is also used for all liquid products that would be unstable to heat or irradiation. Bacteria are removed from the product by ultra fine filtration and hence the product will not be adversely affected. The drawback associated with this method is that the membranes do not remove viral particles, although repeated filtration through 0.1μ membranes can be more effective in this regard. Viral particles can be removed by ultra filtration.
Manufacturing Area
In order to construct a sterile, pharmaceutical product such as this, a manufacturing area will be required. In this case a clean room should be used. A clean room is a working area where the environment is controlled to minimise contamination, and where the sources of contamination are controlled.
Clean rooms are given certain grades depending on what there intended purpose is. The class of clean room depends most importantly on the number of particles per unit volume of air, usually this is the number between 0.5 – 1 μm per cubic metre.
Clean rooms are very expensive to build so it is important to identify what type of contamination you are trying to control and deign accordingly. Since we are dealing with a vaccine the type of contamination that we are primarily concerned about is bacterial contamination. It should also be noted that finished product sterility testing of such preparations represents one of the most critical product tests undertaken by Quality Control. Considering this, we should be using a high class of clean room.
One type of clean room that may be considered is a High Efficiency Particulate Air (HEPA) filter. These filters are the most common type and will remove 99.97% of 0.3 μm particles. HEPA filters are made from fibreglass and they work because small particles will stick to the fibres as they are blown through. HEPA filters are fragile and should be tested for leaks after installation. A HEPA filter will not remove all the particles from a room. Larger pieces of dirt will settle onto surfaces and then must be removed by cleaning or vacuuming. ULPA (Ultra Low Penetration Air) filters could also be used. They have a greater efficiency, removing 99.999% of 0.3 μm particles, and are used mainly in high quality clean rooms.
When dealing with clean rooms a large amount of monitoring is required. There should be a pressure differential between a clean room and the outside areas in order to ensure that dirty air does not pass into the clean room. The use of barometers can check this pressure difference.
Static electricity must be considered since it will cause accumulation of dirt on surfaces designed to be clean and can even cause damage to electronics by electrostatic discharge. There are many ways to assess static in a clean room, these include air ion counters, static fieldmeters, charge plate monitors and ion balance meters.
In a sterile environment, the air must be checked regularly to see how many bacteria and fungi spores are present. It is also necessary to identify the species found. Types of bacteria are easily identified from the colony morphology and microscopic appearance. It is important to observe any changes in the pattern of contamination as this may indicate a new source of contamination.
Finally, when using a clean room it is imperative that the correct type of protective clothing is worn, since the people using the clean room are the biggest source of contamination.
In general, gloves are normally worn, the choice of which depends on the material being handled. Powdered gloves should not be used. Head covers should cover all of the hair, and should be able to contain loose hairs. When hoods are worn they should be tucked into the coveralls and beard covers should always be worn. Body covers are usually suits that cover the entire body and should contain all particles that are shed form the body. Foot cover is vital in preventing dirt entering a clean area, and when working with sterile products facemasks are required. Care should be taken that the masks are changed regularly and they are not lifted out of the way for talking.
Clean room garments should never be worn outside the clean room and outside clothes should never be worn in a clean room, except of course in emergencies.
