Preclinical assessment of Myozyme

Myozyme (tradename) is a recombinant form of the human lysosomal enzyme acid a-glucosidase (GAA) produced in a CHO cell line. It catalyses the degradation of lysosomal glycogen and it is approved for the treatment of Pompe disease (glycogen storage disease type II), an inherited disorder of glycogen metabolism caused by the absence or marked deficiency of lysosomal GAA. The product is administered by i.v. infusion at a dosage rate of 20 mg per kilogram body weight once every 2 weeks. The enzyme is taken up by various cells via endocytosis, triggered by binding of its carbohydrate component to cell surface sugar receptors. Internalization of endocytotic vesicles is followed by fusion with (and hence delivery to) lysosomes.

During product development, many of the initial non-clinical studies were undertaken in GAA knockout mice (i.e. mice devoid of a functional GAA gene), which serves as an animal model for Pompe's disease. The mice proved useful in assessing the pharmacodynamic effect of Myozyme on glycogen depletion and helped establish appropriate dosage regimens. The mice were also used to evaluate pharmacokinetics and biodistribution of GAA following its administration at clinically relevant doses.

Initial safety tests were carried out in beagle dogs and subsequently in cynomolgus monkeys. Single bolus i.v. doses of up to 100 mg kg-1 were used and were found to exert no negative effect upon general condition, blood pressure, heart or cardiovascular parameters, respiration rate or body temperature. No safety tests evaluating potential product effects upon the central nervous system were undertaken, as the protein is considered unlikely to cross the blood-brain barrier.

Repeat dose pharmacokinetic studies were undertaken in Sprague-Dawley rats and in monkeys. Biodistribution studies were carried out in both normal and knockout mice, with the majority of product distributed to the liver. No specific studies on product metabolism or excretion were undertaken, as the protein is almost certainly degraded via normal protein degradation mechanisms.

Toxicity was evaluated in mice, rats, dogs and monkeys following both acute and chronic product administration at various dosage levels (1-200 mg kg-1 range), with a proportion of animals displaying hypersensitivity/anaphylactic-like responses at high dosage levels.

Genotoxicity studies were not undertaken; this is normal practice for protein-based biophar-maceuticals, as proteins are unlikely to have mutagenic potential. No carcinogenicity studies were undertaken. Such studies are not normally required for therapeutic proteins unless there is some specific concern about carcinogenic potential. Reproductive toxicity studies evaluating product effect upon embryo-foetal development were undertaken in mice and revealed no concerns. Product antigenicity was evaluated, mainly as part of chronic toxicity studies. Antibody production against product was evaluated over a 26-week repeat administration period in monkeys. An enzyme-linked immunosorbent assay (ELISA)-based immunoassay (Chapter 7) was used to detect and quantify anti-product antibodies, with all monkey studies developing such antibodies.

Table 4.4 The clinical trial process. A drug must satisfactorily complete each phase before it enters the next phase. Note that the average duration listed here relates mainly to traditional chemical-based drugs. For biopharmaceuticals, the cumulative duration of all clinical trials is, on average, under 4 years

Table 4.4 The clinical trial process. A drug must satisfactorily complete each phase before it enters the next phase. Note that the average duration listed here relates mainly to traditional chemical-based drugs. For biopharmaceuticals, the cumulative duration of all clinical trials is, on average, under 4 years

Trial phase

Evaluation undertaken (and usual number of patients)

Average duration (years)


Safety testing in healthy human volunteers (20-80)



Efficacy and safety testing in small number of patients (100-300)



Large-scale efficacy and safety testing in substantial numbers of


patients (1000-3000)


Post-marketing safety surveillance undertaken for some drugs that


are administered over particularly long periods of time (number of

patients varies)

• the toxicological properties of the drug in humans (with establishment of the maximally tolerated dose);

• the appropriate route and frequency of administration of the drug to humans.

Thus, the emphasis of phase I trials largely remains upon assessing drug safety. If satisfactory results are obtained during phase I studies, the drug then enters phase II trials. These studies aim to assess both the safety and effectiveness of the drug when administered to volunteer patients (i.e. persons suffering from the condition the drug claims to cure/alleviate).

The design of phase II trials is influenced by the phase I results. Phase II studies typically last for anything up to 2 years, with anywhere between a few dozen and a hundred or more patients participating, depending upon the trial size.

If the drug proves safe and effective, phase III trials are initiated. (In the context of clinical trials, safe and effective are rarely used in the absolute sense. 'Safe' generally refers to a favourable risk:benefit ratio, i.e. the benefits should outweigh any associated risk. A drug is rarely 100 per cent effective in all patients. Thus, an acceptable level of efficacy must be defined, ideally prior to trial commencement. Depending upon the trial context, 'efficacy' could be defined as prevention of death/prolonging of life by a specific time-frame. It could also be defined as alleviation of disease symptoms or enhancement of the quality of life of sufferers (often difficult parameters to measure objectively). An acceptable incidence of efficacy should also be defined (particularly for phase II and III trials), e.g. the drug should be efficacious in, say, 25 per cent of all patients. If the observed incidence is below the minimal acceptable level, then clinical trials are normally terminated.

Phase III clinical trials are designed to assess the safety and efficacy characteristics of a drug in greater detail. Depending upon the trial size, usually hundreds if not thousands of patients are recruited, and the trial may last for up to 3 years. These trials serve to assess the potential role of the new drug in routine clinical practice; the phase III results will largely dictate whether or not the prospective drug subsequently gains approval for general medical use.

Even if a product gains marketing approval (on average, 10-20 per cent of prospective drugs that enter clinical trials are eventually commercialized), the regulatory authorities may demand further post-marketing surveillance studies. These are often termed 'phase IV clinical trials'. They aim to assess the long-term safety of a drug, particularly if the drug is administered to

Figure 4.9 Scale-up of proposed biopharmaceutical production process to generate clinical trial material, and eventually commercial product. No substantive changes should be introduced to the production protocol during scale-up

patients for periods of time longer than the phase III clinical trials. The discovery of more long-term unexpected side effects can result in subsequent withdrawal of the product from the market.

Both preclinical and clinical trials are underpinned by a necessity to produce sufficient quantities of the prospective drug for its evaluation. Depending on the biopharmaceutical product, this could require from several hundred grams to over a kilogram of active ingredient. Typical production protocols for biopharmaceutical products are outlined in detail in Chapter 6. It is important that a suitable production process be designed prior to commencement of preclinical trials, that the process is amenable to scaling up and that, as far as is practicable, it is optimized (Figure 4.9). The material used for preclinical and clinical trials should be produced using the same process by which it is intended to undertake final-scale commercial manufacture. Extensive early development work is thus essential. Any significant deviation from the production protocol used to generate the trial material could invalidate all the clinical trial results with respect to the proposed commercialized product. (Changes in the production process could potentially change the final product characteristics, both for the active ingredient and the contaminant profile.)

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