Ethical and Scientific Perspectives of Placebo-controlled Trials in Schizophrenia
2009

Yuval Melamed
Adiel Doron
Orit Stein-Reisner
Avi Bleich
Ethical and Scientific Perspectives of Placebo-controlled Trials in Schizophrenia

J Clin Med Res •
13200910.4021/jocmr2009.07.1247Review Press Elmer ArticlesPlacebo-controlSchizophreniaMedical ethicsClini- cal trials
Clinical trials for development of new medications are essential in all fields of medicine. The requirement for a placebo arm in pharmaceutical trials presents ethical and clinical dilemmas that are especially complicated with regard to mentally ill persons whose free choice and ability to provide informed consent may be questionable. On the other hand, we do not believe that this predicament justifies unconditional rejection of placebo use in psychiatry, when the investigational drug may ultimately provide substantial benefit for some patients. At the same time it is the psychiatrist's responsibility to insure that investigators are adequately trained to conduct clinical trials and that stringent regulatory committees supervise the scientific, clinical and ethical aspects of the trials.

Introduction

Clinical trials involving human subjects give rise to ethical and medico-legal dilemmas. Essential research of new drugs may potentially expose patients to ineffective medications or to placebo. The complexity of the issue increases when dealing with mentally ill patients for whom there is no known cure, and whose ability to provide informed consent to participate in clinical trials may be questionable.

In the absence of a consensus regarding criteria for placebo use Kotler and Witztum [1] claim that we are witnessing a growing controversy. This debate has practical implications for Ethics Committees (Internal Review Boards) when deliberating approval of participation in extensive essential psychopharmacological research. The issue has been addressed by psychiatrists in various countries [2]. We present the ethical considerations to be dealt with by the treating physicians and investigators.

Declaration of Helsinki

The basic principle of the Declaration is that each and every patient is entitled to the best treatment available [3]. In 1996 it was determined that in all medical research, every patient, including those in control groups is entitled to the best available treatment, and that placebo may be used only in the event that there is no proven treatment available.

In the year 2000 a correction was added, which determined that the benefits, risks and effectiveness of new treatments must be weighed in comparison to the very best available treatment, and the use of a placebo arm should be limited to situations where there is no proven effective treatment.

In 2002 the World Medical Association (WMA) inserted a clarification that claimed that placebo-controlled trials could be ethical, even where proven treatments are available in the event that: there are scientific/methodological grounds for inclusion of placebo in the trial as an essential condition to prove efficacy of the drug [4].

Research versus treatment

Medical treatment and medical research differ. The Physician's Oath, adopted by the General Assembly of the World Medical Association, (1948, amended 1968) pledges that "The health of my patient will be my first consideration". Naturalistic or case control study designs do not interfere with the patients' treatment regimens, and they are in accord with these principles of treatment. However investigations of new drugs are randomized controlled trials which require research methods that often contradict accepted standards of treatment.

Determination of dosage is in accordance with the research protocol, and may be influenced by additional agents including investigators, medical institutions, commercial and insurance companies [5].

An additional problem is that clinical trials often require a washout period during which the patient receives no effective treatment.

Types of studies

A number of study designs can be used to prove treatment efficacy: placebo-controlled trials; active-drug comparison trials, control group in comparison of dosages, historical control groups and control groups with no treatment [6]. The latter two designs are not considered valid for psychopharmacological studies.

In placebo-controlled studies, or dosage comparisons, the goal is to emphasize superiority of the new drug, in any dosage, in comparison to placebo, or a lower dosage of the same drug.

In a trial with a control group receiving active treatment -the aim is to prove superiority of the new drug over the active comparator. It is difficult to expect a new treatment to be better than a recognized standard therapy during the various stages of the study, therefore many investigators would prefer the situation where studies that plan to show equal value or non-inferiority would be considered valid.

The research hypothesis that it is possible to differentiate between various treatments is called assay-sensitivity. This assumption is based on prior studies with active standard treatments, but it should be noted that the instruments used for measurement in psychiatry are often based on observation, self-report or interview and not on objective measures. Some medications that are in use and considered effective, have not always been proven as such in comparison to placebo. Thus, if we compare a new drug to these agents, we cannot scientifically prove that the new drug is effective, even if the trial reveals that it is of equal value to existing medications.

A placebo arm in a clinical trial enables investigation of assay sensitivity. When there is no placebo arm in a trial, it may be necessary to increase the size of the study group to prove the postulation of non-inferiority, thus complicating performance of the study. In addition, in such a case more patients will be exposed to an experimental drug whose efficacy has yet to be proven. Similarly, the smaller the gap, when comparing new and existing treatments, the larger the study sample needed in order for the difference to have statistical significance.

An additional experimental possibility which is used, for example in cardiology, is an add-on trial drug with a placebo-control [7]. This method (placebo-controlled addon) reduces the dilemma of administering placebo, since all participants receive active treatment.

Informed Consent

Due to the potential discordance between research and treatment, the patient must be aware that he/she is being treated in the framework of a research protocol and he/she must provide written informed consent. Since the competency of schizophrenia patients to provide valid informed consent is questionable, stringent regulation is essential for the informed consent process involving this population.

Thought disturbances and cognitive impairments characteristic of schizophrenia may give rise to concern regarding the ability of the patient to provide informed consent [8] especially patients with chronic illness [9].

In addition, the psychiatric patient who is hospitalized for long periods of time may more readily agree to participate in clinical trials offered by his attending physician, because of his/her dependency on the hospital.

The Working Party on Research on the Mentally Incapacitated, London, discussed the issue [10] and determined that many people with mental impairment or disorders are able to consent to their inclusion in research provided care is taken to explain it to them. Patient's competence should be evaluated using questionnaires designed especially for that purpose [11]. However, there are those who believe that provision of informed consent neither protects the patient nor ensures an ethical basis for the study since the patient's condition interferes with his/her ability to objectively weigh the various treatment options [12].

Placebo controlled trials in schizophrenia

The need for new pharmacological treatments is based on the fact that some patients respond to currently available antipsychotic agents with only partial improvement, especially in all aspects related to negative schizophrenia symptoms. The existing treatment for this disease is effective mainly for positive symptoms and in delaying relapse of the disease in chronic patients. Moreover, the efficacy of antipsychotics in preserving cognitive functions has yet to be proven. In addition, the existing drugs, including second generation medications, have significant side-effects that may impair the health and quality of life of schizophrenia patients. In the formal sense there is no Gold Standard of care for negative symptoms, so it is necessary to engage in research that compares efficacy of the preparation to placebo [13].

In the United States, to gain Food and Drug Administration (FDA) approval, significant proof of the efficacy of the drug based on adequate controlled trials must be presented [14]. The majority of investigative new drug applications is based on the results of placebo-controlled trials [15]. The European authority parallel to the FDA approves drugs based on trials where the new preparation is compared to placebo and/or treatment with standard medication.

Positive response to placebo in schizophrenia has been shown to range from 3 to 26% in various studies [16]. Different patients respond differently to placebo, including those who apparently respond better to placebo, and it is probably not worthwhile to include those patients, if it is possible to identify them ahead of time [17].

Difficulties regarding placebo control in schizophrenia and possible solutions

The main argument against placebo use is associated with the inherent difficulty of the physician to refrain from giving the patient effective treatment. This issue is difficult for every physician/investigator and especially for physicians who treat the mentally ill.

Absence of active treatment may cause long and shortterm risk or harm. Early diagnosis and treatment has been shown to achieve the best possible results in reducing negative symptoms [18] or improving the chance for a longer remission [19]. Although some studies showed that even if the patients suffered exacerbation of illness after stopping active treatment, they returned to their basic state within a number of days or weeks with no long-term harm [20], however not all patients enter remission following episodes of exacerbation.

Thus, we believe that first episode patients should not be recruited to clinical drug trials unless the study focuses on treatment specific to this population.

The emotional state of the patients and the possibility of cognitive impairment raise questions regarding their capacity to provide informed consent for participation in research. This can be avoided by routinely using questionnaires that specifically examine the patient's competency to participate in clinical trials [11]. In order to eliminate staff influence, the research staff should be distinguished from the caregiving staff, and an external physician may participate in the recruitment stages of the investigation.

Patients receiving involuntary treatment either in the hospital or in the community require special consideration regarding recruitment to placebo-controlled trials. It is questionable whether a patient who does not consent to hospitalization can voluntarily consent to participation in a pharmaceutical trial. Alternatively, there is the view that though a patient may oppose treatment and hospitalization, if medication is compulsory, he/she may prefer one treatment over another, or may agree to a specific treatment [2]. The authors believe that these patients should not be included in clinical trials, due to the sensitivity of the issue.

Other opinions suggest that patients recruited to placebo studies are not necessarily representative of the general patient population. They may be older, with a longer duration of illness and more willing to participate in clinical trials because a cure has not yet been found for their illness. In addition, Placebo-controlled trials have a higher dropout rate [21]. A significant problem in commercial studies is the financial aspect: Today, most clinical trials for new drug applications are designed and sponsored by pharmaceutical companies. Commercial companies are interested in placebo-controlled trials and the direct financial relationship between the company and the investigator may be problematic [22], and should be avoided.

The investigator must assure beneficence, a favorable balance between the potential benefit and harm of participation.

Discussion

There is no known pharmacological cure for schizophrenia. Treatment provided by physicians is in accord with the current knowledge of the illness, and is dispensed to alleviate the symptoms and to improve the patient's ability to function, and quality of life. Physicians will always hesitate to expose a patient to an unfamiliar agent when a known medication is obtainable. On the other hand, without medical research and clinical trials, we would not have the effective medications that we have today, which pending ongoing medical research, may indeed be replaced by more effective preparations in the future.

The increasing rift surrounding the use of placebo in trials for the development of new psychiatric medications, involves scientific, clinical and ethical issues. It seems that there is no single all encompassing approach either for or against placebo-controlled trials. Placebo use is problematic in medicine. Administering treatment which in fact may be a placebo alters the physician-patient relationship, especially when dealing with mental health patients suffering from schizophrenia. However, since there is no known cure for the disease, research is essential in this population, so it may be justifiable to encourage patient participation in clinical trials to advance medical knowledge in the field. In most cases the use of an active control in schizophrenia research is ethically and scientifically preferable [23]. This is not always possible.

It is the investigator's responsibility to ensure that clinical trials are designed and conducted according to appropriate clinical and ethical principles and that each proposed trial is based on solid hypothesis according to which the new treatment will be more effective and/or have less side-effects than existing treatment.

In a clinically justified trial, when including a placebo control group, the size of the placebo group and duration of administration of placebo should be as limited as possible and the duration of the medication washout period should be as short as possible.

A balanced approach should allow for placebo use while examining the various aspects and delineating safeguarding restrictions accordingly [24]. The investigator should bear in mind that referring a patient to participate in a study is basically positive.

It should be emphasized that participation in clinical studies has benefits for the patient, such as increased monitoring and clinical care in the research setting where he/she is examined more frequently, intensified psycho-social services, and the opportunity to continue to receive the experimental drug free of charge following successful completion of the study.

Active quality control of clinical trials should be carried out by the trial sponsor or by their representatives [25]. Quality control begins from the stage of the presentation of the study protocol to the potential investigator for a feasibility review, and is complete when conclusions are drawn and findings are reported.

It is important to train young investigators in the methodology and guidelines for good clinical practice for management of clinical trials, with emphasis on the special issues involving psychiatric patients. Conducting trials in accordance with the International Conference of Harmonization (ICH/GCP) Harmonized Tripartite Guidelines for Good Clinical Practice [26] provides assurance of quality research and patient protection. In addition, Internal Review Boards and ethics committees are responsible for safeguarding the rights, safety and well-being of all trial subjects, with special attention to trials that may include vulnerable subjects.

Conclusions

Clinical pharmaceutical trials are essential for the development of new medications in all fields of medicine, including psychiatry. The requirement for a placebo arm in pharmaceutical trials presents ethical and clinical dilemmas that are especially complicated with regard to mentally ill persons whose free choice and ability to provide informed consent may be questionable. The authors do not believe that this predicament justifies unconditional rejection of placebo use in psychiatry. Each case should be considered and evaluated for risks and benefits for participants and for the general patient population involved.

It is therefore important to require adequate training and certification for investigators and to provide stringent restrictions that will enable strict supervision over the scientific, clinical and ethical aspects of clinical trials.

Manuscript accepted for publication June 30, 2009 Lev-Hasharon Mental Health Center, Netanya, Israel, affiliated to the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel b Corresponding author: Deputy Director, Lev Hasharon Mental Health Center. P.O.Box 90000, Netanya 42100, Israel. E-mail: ymelamed@post. tau.ac.il doi:10.4021/jocmr2009.07.1247a

Articles © The authors, Journal compilation © J Clin Med Res and Elmer Press™, www.jocmr.org
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Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs
2001

Pavel Soucek firstname.lastname@example.com
Group of Biotransformations
Center for Occupational Diseases
National Institute of Public Health
Srobarova 48, Praha 10, 100 42Czech Republic

Roman Zuber firstname.lastname@example.com
Department of Pharmacology
Faculty of Medicine
Palacký University
Hnevotinska 3775 15Olomouc

Czech Republic

Department of Medical Chemistry and Biochemistry
Faculty of Medicine
Palacký University
Hnevotinska 3775 15OlomoucCzech Republic

Eva Anzenbacherová evaanzenbacherováemail@example.com
Department of Medical Chemistry and Biochemistry
Faculty of Medicine
Palacký University
Hnevotinska 3775 15OlomoucCzech Republic

Pavel Anzenbacher
Department of Pharmacology
Faculty of Medicine
Palacký University
Hnevotinska 3775 15Olomouc

Czech Republic

F Peter Guengerich
Department of Biochemistry and Center in Molecular Toxicology
Vanderbilt University Medical Center
37232-0146NashvilleTNUSA

BMC Pharmacology

Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs

BMC Pharmacology
1112001BioMed Central Research article *Corresponding author
Background:The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs.Results:The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the Nterminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4.Conclusions:Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.

Background

Cytochromes P450 (EC 188.8.131.52, CYP) enzymes are known to metabolize the majority of drugs, to detoxify environmental pollutants as well as to activate some classes of carcinogens as polycyclic aromatic hydrocarbons or nitrosamines [1][2][3].

Detoxifying systems of minipig and pig liver have recently attracted considerable attention [4][5][6][7][8] as the minipig and pig liver and hepatocytes are believed to be a possible solution for construction of bioartificial liver devices designed to overcome the shortage of human organs for transplantation [7,9]. Moreover, pigs and especially minipigs might be good model species for general studies in pharmacology and toxicology without the need to induce biotransformation enzymes [10]. Minipig and pig liver have been shown to express the main biotransformation enzymes in amounts and activities comparable to their human counterparts [4,[6][7][8]11]. Three cDNA clones from a porcine small intestine cDNA library were identified as transcripts of three members of porcine CYP gene subfamily, CYP2D25, CYP3A29, and truncated CYP2C42; the fourth cDNA clone appeared to encode a putative CYP2C pseudogene [12,13].

An advantage of the minipig is that it is apparently close to the conventional pig and, hence, the properties of the drug metabolizing systems should be very similar. In our previous work, we have found CYP1A, 2A, 2C, 2D, 2E, and 3A marker activities in minipig liver microsomes by testing the respective specific substrates for human CYP enzymes [4]. In this paper, we report the results of the isolation and characterisation of the first three CYP enzymes from minipig liver microsomal fraction belonging according to their activities as well as to their N-terminal amino acid residues to the CYP2A, CYP2C, and CYP3A subfamilies.

Results and discussion

The microsomal fraction of minipig liver homogenate has been shown to contain the activities characteristic of human CYP3A4 (nifedipine oxidation), 2A6 (coumarin 7-hydroxylation), 2D6 (bufuralol 1'-hydroxylation), 2E1 (p-nitrophenol hydroxylation), and 2C9 (tolbutamide hydroxylation) [4]. The presence of these CYP enzymes in minipig microsomes was confirmed by immunoblotting using antibodies against the respective human P450 enzymes (results not shown). These results have confirmed our earlier finding [4] as well as the results of other authors on pig [6,7,14] and minipig liver microsomal systems [8,15].

The isolation procedure led to fractions containing partially purified CYP enzymes in which the specific activities of CYP3A and 2A forms were detected, the activity of the CYP2C enzyme in the partially purified sample was not estimated as it has been consumed by experiments aimed at characterisation of selected proteins. The main purpose of this work was to obtain partially purified preparations, confirm the presence of selected CYP protein forms in them and with help of electrophoresis and immunoblotting to detect the corresponding CYP proteins. Subsequently, to use the identified CYP forms for N-terminal protein sequencing. Specific minipig CYP enzymes in the enriched fractions were visualised by immunostaining with cross-reacting polyclonal antibodies raised against particular human CYP enzymes (Fig.  1A,B,C). The bands on the blots stained for total protein by Coomassie Blue R-250 corresponding by their position to migration of purified human isoforms and yielding also the bands by immunostaining, in other words, identified this way to correspond to the respective human enzyme, were excised and subjected to N-terminal sequencing. This approach allowed identification of the first 20 amino acids from N-terminus of minipig CYP corresponding to human CYP2A6, 18 amino acids from N-terminus of minipig CYP corresponding to human CYP2C9, and 20 amino acids from N-terminus of minipig CYP corresponding to human CYP3A4. N-Terminal amino acid sequences or 5'-end cDNA sequences of pig or minipig CYP2A and CYP2C have not been published to date, therefore, we compared our results directly with the known human CYP2A6 and 2C9 sequences (Table 1).

From this comparison it can be concluded that the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (14 of 20 amino acids identical). Comparison of CYP2C family members may be quite difficult because of very high content of Leu at the N-terminus of the sequenced minipig protein (still about 50% sequence identity with the human counterpart, Table 1). Moreover, the human CYP2C subfamily has at least four highly homologous clones (sequence identity at the N-terminus > 90%), and therefore the existence of other CYP2C-related genes in the minipig may be anticipated. This problem may hamper further attempts to identify minipig CYP2C proteins with the approach we have used. cDNA cloning should help to answer this question much better.

The protein sequence of another CYP protein present in the sample matched very well the N-terminal amino acid sequence deduced from published cDNA for pig CYP3A29 (Table 1, data from [13]). The pig/minipig CYP3A and human CYP3A4 shared about 60% sequence similarity (12 of 20 amino acids identical). The presence of a minipig liver CYP3A enzyme with similar activities to the human CYP3A4 has been reported earlier [4]. Together with the data obtained with pig liver and intestinal microsomal systems [16][17][18], the results support the suitability of pigs/minipigs for modeling the biotransformation of drugs in man.

From our results it seems that pigs and minipigs have CYP2A, 2C, and 3A liver microsomal enzymes with very similar N-terminal sequences to the human enzymes. This finding may be important for pharmacological and toxicological studies because i) CYP3A, 2C and 2A enzymes metabolize many known industrial chemicals and drugs in human use and ii) minipigs in pharmacology/ toxicology are much easier to handle than conventional pigs as model animals. The observed high similarities of N-terminal sequences of minipig and human CYP2A and 2C confirm the previously published similarity in marker activities [4,8].

Conclusions

The results presented in this study support the use of minipigs as experimental animals to predict biotransformation pathways in man and should stimulate further research on similarity of structure and substrate specificity of individual human and (mini)pig CYP enzymes. The conclusions then obtained may in future bring justification for the use of pig hepatocytes and liver for extracorporeal detoxification and xenotransplantation.

Materials and methods

Chemicals

All reagents and chromatographic materials were purchased from Sigma-Aldrich (Prague, CR) if not stated otherwise and were of the analytical grade purity. DEAE Sephacel was product of Pharmacia Biotech (Uppsala, Sweden). Polyclonal rabbit anti-CYP2A6, anti-CYP2C9, and anti-CYP3A4 IgG were prepared as described elsewhere [19]. Human recombinant CYP2A6, 2C9, and 3A4 were expressed in Escherichia coli and purified as described previously [20][21][22] and used as standards.

Purification of minipig enzymes

Microsomal fractions of liver homogenates were prepared from minipig livers (Brno white variety of Goettingen minipig, Research Institute of Veterinary Medicine, Brno, CR, 25-30 kg body weight, male castrates, N = 5, age 6 months). No induction protocols were applied to minipigs. The preparation of microsomes was done according to standard procedure [23]. Separation of CYP enzymes from cholate-solubilized microsomal fraction was based in general on the procedure developed earlier [24,25]. Solubilized microsomes were applied to an octyl-Sepharose column where the NADPH-cytochrome P450 reductase was eluted first during the wash with
1 1 0 2 0 Minipig CYP2A M L A S G L L L V A L L T/L R L X I F V L Human CYP2A6 a M L A S G ML L V A L L V C L T V M V L 1 1 0 1 8 Minipig CYP2C M D V L V X L A L X L L L V X L L L Human CYP2C9 b M D S L V V L V L C L S C L L L L S 1 1 0 2 0 Minipig CYP3A

M D L I P G F S T E T W V L L A T S L V Pig CYP3A29 c M D L I P G F S T E T W V L L A T S L V Human CYP3A d M A L I P D L A M E T R L L L A V S L V

Notes: X -amino acid was not identified. Similarities between pig and minipig cDNAs in bold. GeneBank accession numbers: a AF182275, b M61855, c Z93099, d AF209389 equilibration buffer (buffer A, 0.1 M K/PO 4 , pH 7.25, 1 mM EDTA, 20% (v/v) glycerol, 0.6% (w/v) cholate), in which the cholate concentration was reduced to 0.42%. A sharp peak containing cytochrome b 5 together with CYP3A was eluted when the concentration of cholate was further lowered to 0.33% and 0.06% (w/v) Triton N-101 was added to buffer A. The next fractions eluted after CYP3A and cytochrome b 5 appeared to contain mainly the CYP2C and CYP2D enzymes. Lastly, the fractions with CYP2A were eluted. CYP3A was separated from cytochrome b 5 by anion exchange chromatography on DEAE Sephacel equilibrated with buffer B: 5 mM K/PO 4 , pH 7.7, containing 0.1 mM EDTA, 20% glycerol (v/v), and 0.2% sodium cholate (w/v). Increasing the cholate concentration to 0.5% along with addition of 0.2% Triton N-101 (w/v) resulted in elution of the CYP containing peak. Impure fractions containing CYP were dialyzed overnight against 10 mM potassium phosphate, pH 7.4, containing 0.05 mM EDTA, 0.1 mM dithiothreitol, and 20% glycerol (v/v) and further purified by chromatography on a hydroxylapatite column equilibrated with the same buffer. Extensive washing was done to remove Triton N-101. CYP was eluted using a linear gradient of phosphate, from 10 mM to 500 mM. The hydroxylapatite chromatography was repeated to further purify the CYP enzyme fractions prior to immunoblotting and amino acid sequencing.

Activity assays

The results of activity assays are displayed in Table 2.

Nifedipine oxidation was followed to characterize the CYP3A enzyme activity according to [26]. Oxidation of nifedipine was determined in microsomes as well as in fractions after chromatography. In this case, a reconstituted system including 200 pmol of NADPH-cytochrome P450 reductase (PanVera, Madison, WI) was used with a procedure described previously [21]. Coumarin 7-hydroxylation was followed to estimate the CYP2A activity [27]. To monitor the activity of CYP2C enzyme(s), in microsomal faction, tolbutamide hydroxylation was determined [28]. The determination of activities in fractions containing partially purified CYP enzymes was done in systems mimicking the original microsomal preparations with addition of components lost during the process of purification of CYP enzymes (NADPH:cytochrome P450 reductase, cytochrome b 5 , phospholipid). The method was based on general approach described by Shimada and Yamazaki [29].

Electrophoresis and immunoblotting

SDS electrophoresis was done in 10% and 8% (w/v) polyacrylamide gels by the method of Laemmli [30] using a MiniProtean apparatus (BioRad, Hercules, CA). Protein staining was done with Coomassie Blue R-250 [31] and immunoblotting was performed as described [32] using the described conditions for development of blots [19]. Polyclonal anti-human CYP2A6, 2C9 and 3A4 IgG were used.

Amino acid sequencing N-Terminal amino acid sequencing was performed using Procise Protein Sequencer (Applied Biosystems, Foster City, CA) and methodology based on Edman degradation. SDS electrophoresis, transfer of protein to Immobilon-P membrane (Millipore Corp. Bedford, MA), and staining methods are described elsewhere [19]. Yields at each cycle were estimated by comparison with external standards. The nifedipine oxidase (CYP3A), coumarin 7-hydroxylation (CYP2A) and tolbutamide methyl-hydroxylation activities in microsomes and fractions containing partially purified CYP enzymes were determined as indicated in Materials and Methods, n.d., not determined.

Figure 1
1Immunoblotting of enriched samples of minipig CYPs Lane 1: 1 pmol of the respective CYP standard; lanes 2 and 3: CYP3A-rich fractions, 5 pmol; lanes 4 and 5: CYP2A-and 2C-rich fractions, 12.5 pmol applied per lane onto 8% gel. Blot development: A -100 µg of anti 2A6 IgG; B -50 µg of anti 2C9 IgG; C -50 µg of anti 3A4 IgG.

Table 1 :
1Comparison of Human, Pig, and Minipig CYP N-terminal sequencesGene
Sequence (amino acid No.)

Table 2 :
2Specific activities of individual CYP enzymes (pmol product/nmolP450/min)Sample
CYP3A
CYP2A
CYP2C

Liver microsomal fraction
2193 ± 523
210 ± 70 136 ± 31
CYP -containing fractions 3716 ± 428
423 ± 148 n.d.

AcknowledgementWe thank E. Howard for technical assistance with the Edman degradation. The financial support from Grant Agency of the Czech Republic (grant 203/ 99/0277), Czech Ministry of Education, Youth and Sports (project MSM 151100003) and United States Public Health Service (grants R35 CA44353 and POI ES00267) is gratefully acknowledged.
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