A foetus is donated by a woman and transported in medium to the hospital (yellow). In the clinic (orange), under responsibility of the practitioner, the CNS is taken from the foetus and the ventral mesencephalon (VM) dissected mechanically. Foetal dopaminergic cells are obtained by enzymatic dissociation, washed and injected into the brain by stereotactic guided transplantation.

This relatively easy concept of a cell therapeutic approach might now be translated into the "new language" after the ATMP-regulation became active. "Easy concept" in this case is generally spoken compared to possible modern or future approaches using for example iPS cells, which have been genetically engineered, cultured in humid incubators or bioreactors for days and further manipulated using growth factors and hydrogels, while the classical and most applied concept of cell therapy for Parkinson's disease generally was mostly composed of cells and tissue fragments, dissected from foetal brain tissue without further cell culture but rather direct transplantation into the brain of a patient.

As to date - to our knowledge - there is no application sent to the EMA regarding this cell therapy approach. There might likely be applications in the near future as the data of the earlier clinical trials are promising but need further refinement which can be achieved via additional pre-clinical studies. These investigations will be confronted with defined expressions like good laboratory practice (GLP), good manufacturing practice (GMP) and good clinical practice (GCP), generally considered good professional practice (GxP) as the ATMP regulation is defining such cell therapies as medicinal products, whose production are further defined commonly in numerous regulations and directives.

#### **3. Translating the regulations into (practical) clinical research**

In general, a new clinical trial in Parkinson's disease in the EU using the cell therapy approach which likely becomes the status of an ATMP, would have to be conducted

enzymatically dissociated in combination with DNAse and finally transplanted

This flow sheet shows schematically the conventional process steps for the production of the

A foetus is donated by a woman and transported in medium to the hospital (yellow). In the clinic (orange), under responsibility of the practitioner, the CNS is taken from the foetus and the ventral mesencephalon (VM) dissected mechanically. Foetal dopaminergic cells are obtained by enzymatic dissociation, washed and injected into the brain by stereotactic

This relatively easy concept of a cell therapeutic approach might now be translated into the "new language" after the ATMP-regulation became active. "Easy concept" in this case is generally spoken compared to possible modern or future approaches using for example iPS cells, which have been genetically engineered, cultured in humid incubators or bioreactors for days and further manipulated using growth factors and hydrogels, while the classical and most applied concept of cell therapy for Parkinson's disease generally was mostly composed of cells and tissue fragments, dissected from foetal brain tissue without further

As to date - to our knowledge - there is no application sent to the EMA regarding this cell therapy approach. There might likely be applications in the near future as the data of the earlier clinical trials are promising but need further refinement which can be achieved via additional pre-clinical studies. These investigations will be confronted with defined expressions like good laboratory practice (GLP), good manufacturing practice (GMP) and good clinical practice (GCP), generally considered good professional practice (GxP) as the ATMP regulation is defining such cell therapies as medicinal products, whose production

In general, a new clinical trial in Parkinson's disease in the EU using the cell therapy approach which likely becomes the status of an ATMP, would have to be conducted

stereotactically into the brain of PD-patients (Figure 4).

Fig. 4. Flow-Sheet of a possible cell therapy for Parkinson's Disease

cell culture but rather direct transplantation into the brain of a patient.

are further defined commonly in numerous regulations and directives.

**3. Translating the regulations into (practical) clinical research**

cell preparation for the previously mentioned clinical studies.

guided transplantation.

following the Clinical Trial Directive (Directive 2001/20/EC) as well as the GCP Directive (Directive 2005/28/EC). While these directives are individually to be implemented at a national level for each Member State and can thereby be interpreted variably, the content for EU wide clinical studies is directly applicable. For the example of cell therapy as a clinical study, the manufacture of test material ("the cells") and the production of scientific data on the safety of the test material are particularly relevant. The GCP-Directive provides, inter alia, that relevant data from preclinical experiments should be planned and conducted by following the standards of good laboratory practice (Directive 2004/10/EC), which means in particular for university facilities a major hurdle as GLP accredited laboratories are rare at such institutes and preclinical data at the university level is usually achieved from via basic research. The logical background to the requirement that certain pre-clinical data shall be generated to the standards of GLP is based on the fact that safety issues of the treatment have been generated by reproducible and fully traceable experiments. GLP also means that the data obtained is reliable and understandable for the authorities; that the data will be archived and regularly monitored; and that audits of the laboratory by the authorities are able to demonstrate sustained and continuous improvement. All aspects of a treatment or manufacturing processes that could result in harm to the patient should therefore be carried out with the best possible method to precisely reduce the risk of the patient. In the field of cell therapy, where the cells are considered an implantable drug, the risks considered are aspects of biocompatibility, toxicity, formulation and use of additives or their removal.

Nevertheless, it is our experience that regulatory authorities generally do follow and control upon the principles defined in these GCP and GLP directives. The future clinical trial application on the cell therapy approach for Parkinson's disease using the mentioned set up of enzymatically dissociated primary foetal cells needs to include experimental data generated according to these principles, for example. In addition, it might also be important for the clinical trial group to include a strategy paper on the requirements for the cell therapy approach once the projects leaves the research phases and is about to enter the therapy phase as at this stage GMP principles are to be followed upon as on the EU level this is directed by Regulation (EC) No 1394/2007.

#### **3.1 PD cell therapy according to GLP and GMP**

We pointed out that new clinical cell therapy approaches for PD very likely are to be conducted following the international principles of GLP and GMP, and maybe implemented in slightly varied national versions. As both GLP/ GMP were originally developed on an industrial level for decades to ensure product quality (and safety), such industrial standards are likely to be difficult to implement into a historically evolved research institution as most universities hospitals are like. Even though these research institutions generally conduct research in a "good" way, most likely recognized and approved by high ranking, peer reviewed journals and experts in the specific field of science, this "good" way is different to the requirements of GLP and GMP. We highlight the most crucial elements of both GLP and GMP for the example of cell therapy mentioned above and we will try to provide information on the possible implications of these principles for a university hospital set-up. Both GMP/ GLP share a main common feature: A handbook or manual. This collection of written documents should describe all aspects of the quality management and quality assurance of the proposed cell therapy, and ensure that all activities needed to conduct the safety studies, or the manufacture of the test equipment is shown in clear instructions (standard operation procedures or SOPs)

Transplantation of Foetal Ventral Mesencephalic Grafts

monitored constantly.

specification delivery for production).

in Parkinson's Disease: A Still Evolving Concept with New Regulatory Challenges 389

category B clean room (EudraLex Volume 4, Annex I). For the class B clean room, strictly defined requirements are in place, which vary significantly from the usual requirements of the operating room. For example, it must be a separated room with restricted access and specifically trained staff. The trained staff of the class B clean room must include a Head of Production and a Head of Quality assurance with separated and defined responsibilities. It should also include a Qualified Person who ensures that each lot was produced and tested in accordance with the guidelines and the specifications set within the QM-Handbook. The responsibilities of and legal requirements for a qualified persons are also defined within national/international directives and regulations and usually include a minimum of 2 years work experience with all processes and within the technical requirements (e.g. class B clean room). Aseptic working conditions of the environment should be ensured by performing online particle counts on volumetric air sampling as well as microbiological monitoring using methods such as settle plates or surface sampling during production. Surface sampling includes the active personnel, which fingers should be tested for microbes after each critical process step and less then 5 microbes / 25cm² in class B environment or >1/25cm² inside the class A workbench should be identified (EudraLex, Volume 4, Annex I). It should also be noted that culture and evaluation of the microbiological sampling plates also need to be performed according to the GLP regulations; for example the incubator should be qualified and

Although these examples cited might be considered as common sense, the actual implementation into a regulatory acceptable level is a very serious and work-intensive task. The authorities' want these investigations not only being performed and analyzed, but definitely want to have ensured that all tests or production processes can be performed reproducibly within the acceptable specifications. On the industrial side, from where the regulations originate from, these requirements under GMP conditions are usually implemented through activities such as qualification and validation. In consideration of the aseptic work environment mentioned in this paragraph, in the academic research environment one would ensure quality by qualifying the class A work bench, for example. This type of qualification is, according to the GMP regulations, being performed by the implementation steps of design qualification (what kind of class A bench is needed and how to purchase), installation qualification (verification of complete delivery and installation according to the specifications), operational qualification (writing of SOP and test of functionality) and finally performance qualification (is the work bench fully capable of

This qualification process, using the bench as the example, shows that a large number of documents and tests must be written and performed on a single device, contributing significantly to the manufacturing effort. It is generally not sufficient to order a laboratory work bench from a supplier and use the device directly to produce a cell therapy product. The qualification of the workbench is rather to ensure that optimal aseptic work environment can actually be made possible from a technical point of view and that the equipment is ideal for the production process. The necessary evidence here is carried out as part of the validation, a definition for which there also are continuously evolving industrial standards available (aka ISO norms) that should be followed upon as part of GMP, as well as GLP. For example, the aseptic preparation within the bench it needs to be proven that aseptic conditions actually are achieved before manufacturing begins. This can for example be performed by a cleaning validation, which provides the proof on the

and will be documented in writing. In fact, both manuals have a large amount of common ground as quality is managed similar; but because of their different application areas strong differences stand out. The specific requirements of the manuals are described in the various directives and should be available separately for both the GLP studies as well as GMP manufacturing. It is easily predictable even from this requirement that modern studies of cell therapy now in addition to the clinical study plan, other, more comprehensive documentation are required.

As mentioned, both manuals need to define the quality management system. In principle, this management describes who is authorizing the work (Chapter "Management"), where the work is being done (Chapter "Application area"), who is performing the work (Chapter "Personnel"), what equipment and material will be used (Chapter "Equipment", "Substances", "Purchase and Storage") and how the quality is being maintained (Chapter "Document control", "Quality Assurance"). These possible subdivisions of the term "quality" have been established internationally and this can be found for Europe in the EN ISO 9000 / 9001. Following these requirements, an organization or, as in the example of cell therapy, the clinical facility, can officially be certified to present to the outside the implementation of a quality management system. Especially for the later production according to GMP, the implementation of this process is very important because the regulatory agencies will require an efficient quality management system for the manufacturing license (Regulation (EC) No 1394/2007).

A clinical director therefore requires the implementation of a quality system "by the book" (EN ISO 9001) as it is required for GLP and GMP. However, the clinical director must stand behind that decision and also self-identify himself as a model to implement the benefits of an effective quality management system. He must accompany objectives defined by the development of the system, reclaim reports on a regular basis, arrange and identify corrective measures and ensure that agreements are kept up to date. It is not enough here to employ appropriate quality personnel essential to obtain a certification; quality must be understood and experienced as such, otherwise the implementation of the system is condemned to failure from the start.

Once the clinical director identified and implemented such quality measures as a crucial part of clinical cell therapy according to the regulations, he now will be confronted with the details of GLP and GMP.

#### **3.2 The good practice in the lab: An introduction to quality assurance**

As mentioned above, now that it is required to have a quality management system in place, it also is required to produce quality. For example, in both GLP and GMP, it needs to be ensured that cell therapy for the animal safety studies or for the clinical study is being performed under aseptic conditions. While for a specialist in cell culture or the surgeon in the operating room this situation is clearly understood and naturally carried out using corresponding measures consistently, the proof of sterility at medicinal drug manufacturing under GLP and GMP conditions is mandatory. In addition, of course, working environments have to be clean and need to fulfil the prescribed requirements for the manufacturing of ATMP sufficiently.

In the given example, all high risk steps of the preparation of the cell product (opening vials, pipetting, etc) must therefore be carried out in a so-called "class A in Class B environment" according to the Directive 2003/94/EC and further defined in EudraLex Volume 4. In general, it means that a conventional safety cabinet (Class A) is installed in a

and will be documented in writing. In fact, both manuals have a large amount of common ground as quality is managed similar; but because of their different application areas strong differences stand out. The specific requirements of the manuals are described in the various directives and should be available separately for both the GLP studies as well as GMP manufacturing. It is easily predictable even from this requirement that modern studies of cell therapy now in addition to the clinical study plan, other, more

As mentioned, both manuals need to define the quality management system. In principle, this management describes who is authorizing the work (Chapter "Management"), where the work is being done (Chapter "Application area"), who is performing the work (Chapter "Personnel"), what equipment and material will be used (Chapter "Equipment", "Substances", "Purchase and Storage") and how the quality is being maintained (Chapter "Document control", "Quality Assurance"). These possible subdivisions of the term "quality" have been established internationally and this can be found for Europe in the EN ISO 9000 / 9001. Following these requirements, an organization or, as in the example of cell therapy, the clinical facility, can officially be certified to present to the outside the implementation of a quality management system. Especially for the later production according to GMP, the implementation of this process is very important because the regulatory agencies will require an efficient quality management system for the

A clinical director therefore requires the implementation of a quality system "by the book" (EN ISO 9001) as it is required for GLP and GMP. However, the clinical director must stand behind that decision and also self-identify himself as a model to implement the benefits of an effective quality management system. He must accompany objectives defined by the development of the system, reclaim reports on a regular basis, arrange and identify corrective measures and ensure that agreements are kept up to date. It is not enough here to employ appropriate quality personnel essential to obtain a certification; quality must be understood and experienced as such, otherwise the implementation of the system is

Once the clinical director identified and implemented such quality measures as a crucial part of clinical cell therapy according to the regulations, he now will be confronted with the

As mentioned above, now that it is required to have a quality management system in place, it also is required to produce quality. For example, in both GLP and GMP, it needs to be ensured that cell therapy for the animal safety studies or for the clinical study is being performed under aseptic conditions. While for a specialist in cell culture or the surgeon in the operating room this situation is clearly understood and naturally carried out using corresponding measures consistently, the proof of sterility at medicinal drug manufacturing under GLP and GMP conditions is mandatory. In addition, of course, working environments have to be clean and need to fulfil the prescribed requirements for the

In the given example, all high risk steps of the preparation of the cell product (opening vials, pipetting, etc) must therefore be carried out in a so-called "class A in Class B environment" according to the Directive 2003/94/EC and further defined in EudraLex Volume 4. In general, it means that a conventional safety cabinet (Class A) is installed in a

**3.2 The good practice in the lab: An introduction to quality assurance**

comprehensive documentation are required.

manufacturing license (Regulation (EC) No 1394/2007).

condemned to failure from the start.

manufacturing of ATMP sufficiently.

details of GLP and GMP.

category B clean room (EudraLex Volume 4, Annex I). For the class B clean room, strictly defined requirements are in place, which vary significantly from the usual requirements of the operating room. For example, it must be a separated room with restricted access and specifically trained staff. The trained staff of the class B clean room must include a Head of Production and a Head of Quality assurance with separated and defined responsibilities. It should also include a Qualified Person who ensures that each lot was produced and tested in accordance with the guidelines and the specifications set within the QM-Handbook. The responsibilities of and legal requirements for a qualified persons are also defined within national/international directives and regulations and usually include a minimum of 2 years work experience with all processes and within the technical requirements (e.g. class B clean room). Aseptic working conditions of the environment should be ensured by performing online particle counts on volumetric air sampling as well as microbiological monitoring using methods such as settle plates or surface sampling during production. Surface sampling includes the active personnel, which fingers should be tested for microbes after each critical process step and less then 5 microbes / 25cm² in class B environment or >1/25cm² inside the class A workbench should be identified (EudraLex, Volume 4, Annex I). It should also be noted that culture and evaluation of the microbiological sampling plates also need to be performed according to the GLP regulations; for example the incubator should be qualified and monitored constantly.

Although these examples cited might be considered as common sense, the actual implementation into a regulatory acceptable level is a very serious and work-intensive task. The authorities' want these investigations not only being performed and analyzed, but definitely want to have ensured that all tests or production processes can be performed reproducibly within the acceptable specifications. On the industrial side, from where the regulations originate from, these requirements under GMP conditions are usually implemented through activities such as qualification and validation. In consideration of the aseptic work environment mentioned in this paragraph, in the academic research environment one would ensure quality by qualifying the class A work bench, for example. This type of qualification is, according to the GMP regulations, being performed by the implementation steps of design qualification (what kind of class A bench is needed and how to purchase), installation qualification (verification of complete delivery and installation according to the specifications), operational qualification (writing of SOP and test of functionality) and finally performance qualification (is the work bench fully capable of specification delivery for production).

This qualification process, using the bench as the example, shows that a large number of documents and tests must be written and performed on a single device, contributing significantly to the manufacturing effort. It is generally not sufficient to order a laboratory work bench from a supplier and use the device directly to produce a cell therapy product. The qualification of the workbench is rather to ensure that optimal aseptic work environment can actually be made possible from a technical point of view and that the equipment is ideal for the production process. The necessary evidence here is carried out as part of the validation, a definition for which there also are continuously evolving industrial standards available (aka ISO norms) that should be followed upon as part of GMP, as well as GLP. For example, the aseptic preparation within the bench it needs to be proven that aseptic conditions actually are achieved before manufacturing begins. This can for example be performed by a cleaning validation, which provides the proof on the

Transplantation of Foetal Ventral Mesencephalic Grafts

conserve resources and optimize the expected outcome.

for university and other academic institutions.

**5. Conclusion**

in Parkinson's Disease: A Still Evolving Concept with New Regulatory Challenges 391

recently clearly and very significantly changed so that the implementation of studies on cell therapy for small clinical facilities and centres quickly reaches the limits of feasibility, when

With the desire of reducing the inconsistencies and variations across centres, a consortium of European based leading clinical and research centres implicated in cell therapy recently have been awarded European Union funding aimed at addressing critical questions in the context of cell therapy in PD, particularly concerning patient selection, tissue preparation, cell delivery, immunosuppression, and off-state dyskinesia. A key objective of the TRANEURO project funded by the FP7 European Commission Health programme is to generate standardized protocols that can serve as a template for all future clinical trials in the cell therapy field including stem cell-based therapies for PD. As part of an international multi-centre clinical study, both preclinical and clinical standards are being defined across the centres in order to generate the best practice approaches investigating the feasibility and clinical efficacy of foetal cell therapy in PD (see also TransEuro homepage http://www.transeuro.org.uk/). Furthermore, the stringent regulatory requirements to implement the standards of GMP and GCP will be addressed by close cooperation between the various centres in particular through joint protocol development and the intensive exchange between the project managers of the different areas to combine synergies to effectively take-on the numerous technical and regulatory hurdles together. By using modern communication media such as web meetings, online training classes, an international project management, but also classic laboratory visits, all elements for manufacturing and the clinical treatment of patients are developed and established, to

The legislature has certainly imposed very high standards of patients' safety in clinical trials, but as illustrated in the previous examples, such standards hereby reached a high level that can only be met with great difficulty by classical research institutions alone. The declared goal of the TRANSEURO consortium is to achieve successful cell therapy for patients suffering from PD, but at the same time to provide the feasibility of implementing a therapy at regulatory acceptable level. It is the authors' opinion that the enhancement of the regulations during the last years is a double-edged sword with a potential to become a sharp "weapon". On one hand it maximizes patient and promotes the standardization of innovative procedures. On the other side, it creates a substantial challenge, both for personal and financial resources, to meet the new regulatory processes and requirements especially

Cell transplantation for PD-patients is a promising therapy, based on the previous preclinical and clinical research. The use of foetal tissue as proposed in the TRANSEURO project holds a number of (ethical) concerns and technical handicaps, but is an ideal candidate to merge basic science and industrial standards to achieve an efficient therapy. This may set the basis of future cell therapeutically approaches with considerably more complex processes and thereby noticeable higher regulatory demands. The worldwide effort working with embryonic stem cells and with induced pluripotent stem cells (iPS) might in the near future generate alternative candidate cells for clinical therapy in PD. However, complicated manufacturing processes involving gene manipulation, advanced cell culture techniques and risk of tumorigenicity will benefit greatly from the currently planned

all the national and international laws and guidelines need to be considered.

basis of scientific methods that both the optimum cleaning procedure, the best method as well as the most suitable chemicals were used to scientifically exclude risks to the cell product by exposure to microbes or to the cleaning procedure itself. It is conceivable to prepare an aseptic work surface to roughly prepare or cut the tissue for later cell preparation steps. In this case, however, it should be proven by scientific evidence that no risk is transmitted by the surface to the tissue or indirectly to the patient by chemical residues left over after the cleaning process, a simple fact but overlooked easily and actually technically difficult to prove.

Using as an example the need for aseptic work environment leading up to clinical treatment, it becomes clear that the preparation for cell therapy under either GLP or GMP purposes is labour intensive, including the generation of several SOPs, performing of several validation tests and filling out forms to collect the data. The validation activities thereby need to be planned very carefully as the clinical director never wants only to focus on single issues of the process but on all aspects of the full manufacturing process. For planning validations in a good practice environment, a so-called validation master plan should thereby be discussed and generated before any validation is actually being performed. This plan is (yet) not mandatory in the regulations, but is one of the first documents a regulator officer will ask for during an audit. It provides information of how the clinical department operates, who is in control of the different validation activities, and how the manufacturing, quality control and personnel are being managed. Main integral part of the validation master plan generally is the previously mentioned qualification procedure, but it also includes activities performed to generate general risk analysis documentation, validation approaches to deal with identified risks and timelines to finish the validation activities.

Finally, it is clear that in addition to the head of production, the Quality Assurance Manager and the Qualified Person, a validation expert should also be present in addition to seamlessly link the areas of manufacturing and quality assurance of the product by adapted validation activities. It also becomes clear that once the proposed cell therapy is defined as a medicinal product by the authorities, the principle investigator of the cell therapy - used to research standards - now is confronted with multiple legal requirements that for newcomers might become irritating and frustrating. A high number of needs are to be fulfilled, not only requiring a tremendous piece of work time to be invested to "get the paper work done", but also technical and personal requirements need to be put in place, most likely coming along with a substantial amount of financial demand. If one considers the usually small numbers of "products" the principle investigator will need to address small clinical trials, these high risk investments will not easily find the internal support needed. Nevertheless, in the long-term, once implemented into a working system, this newly implemented system will lead to less hassles and burdens to carry out additional cell therapy studies and significantly promote the standardisation in multicenter trials.

#### **4. Standardizing new cell therapies for PD: The TRANSEURO project**

As discussed in the introduction of this chapter, in recent decades several clinical studies using cell therapy in PD were investigated often producing large differences in the outcome (Winkler, Kirik & Björklund 2005h). In many cases it was found that the protocols needed to conduct the therapy varied significantly in many places and a generalized statement about the efficiency of the studies was difficult to reach. In parallel, the legislation has only recently clearly and very significantly changed so that the implementation of studies on cell therapy for small clinical facilities and centres quickly reaches the limits of feasibility, when all the national and international laws and guidelines need to be considered.

With the desire of reducing the inconsistencies and variations across centres, a consortium of European based leading clinical and research centres implicated in cell therapy recently have been awarded European Union funding aimed at addressing critical questions in the context of cell therapy in PD, particularly concerning patient selection, tissue preparation, cell delivery, immunosuppression, and off-state dyskinesia. A key objective of the TRANEURO project funded by the FP7 European Commission Health programme is to generate standardized protocols that can serve as a template for all future clinical trials in the cell therapy field including stem cell-based therapies for PD. As part of an international multi-centre clinical study, both preclinical and clinical standards are being defined across the centres in order to generate the best practice approaches investigating the feasibility and clinical efficacy of foetal cell therapy in PD (see also TransEuro homepage http://www.transeuro.org.uk/). Furthermore, the stringent regulatory requirements to implement the standards of GMP and GCP will be addressed by close cooperation between the various centres in particular through joint protocol development and the intensive exchange between the project managers of the different areas to combine synergies to effectively take-on the numerous technical and regulatory hurdles together. By using modern communication media such as web meetings, online training classes, an international project management, but also classic laboratory visits, all elements for manufacturing and the clinical treatment of patients are developed and established, to conserve resources and optimize the expected outcome.

The legislature has certainly imposed very high standards of patients' safety in clinical trials, but as illustrated in the previous examples, such standards hereby reached a high level that can only be met with great difficulty by classical research institutions alone. The declared goal of the TRANSEURO consortium is to achieve successful cell therapy for patients suffering from PD, but at the same time to provide the feasibility of implementing a therapy at regulatory acceptable level. It is the authors' opinion that the enhancement of the regulations during the last years is a double-edged sword with a potential to become a sharp "weapon". On one hand it maximizes patient and promotes the standardization of innovative procedures. On the other side, it creates a substantial challenge, both for personal and financial resources, to meet the new regulatory processes and requirements especially for university and other academic institutions.

#### **5. Conclusion**

390 Towards New Therapies for Parkinson's Disease

basis of scientific methods that both the optimum cleaning procedure, the best method as well as the most suitable chemicals were used to scientifically exclude risks to the cell product by exposure to microbes or to the cleaning procedure itself. It is conceivable to prepare an aseptic work surface to roughly prepare or cut the tissue for later cell preparation steps. In this case, however, it should be proven by scientific evidence that no risk is transmitted by the surface to the tissue or indirectly to the patient by chemical residues left over after the cleaning process, a simple fact but overlooked easily and

Using as an example the need for aseptic work environment leading up to clinical treatment, it becomes clear that the preparation for cell therapy under either GLP or GMP purposes is labour intensive, including the generation of several SOPs, performing of several validation tests and filling out forms to collect the data. The validation activities thereby need to be planned very carefully as the clinical director never wants only to focus on single issues of the process but on all aspects of the full manufacturing process. For planning validations in a good practice environment, a so-called validation master plan should thereby be discussed and generated before any validation is actually being performed. This plan is (yet) not mandatory in the regulations, but is one of the first documents a regulator officer will ask for during an audit. It provides information of how the clinical department operates, who is in control of the different validation activities, and how the manufacturing, quality control and personnel are being managed. Main integral part of the validation master plan generally is the previously mentioned qualification procedure, but it also includes activities performed to generate general risk analysis documentation, validation approaches to deal with identified risks and timelines

Finally, it is clear that in addition to the head of production, the Quality Assurance Manager and the Qualified Person, a validation expert should also be present in addition to seamlessly link the areas of manufacturing and quality assurance of the product by adapted validation activities. It also becomes clear that once the proposed cell therapy is defined as a medicinal product by the authorities, the principle investigator of the cell therapy - used to research standards - now is confronted with multiple legal requirements that for newcomers might become irritating and frustrating. A high number of needs are to be fulfilled, not only requiring a tremendous piece of work time to be invested to "get the paper work done", but also technical and personal requirements need to be put in place, most likely coming along with a substantial amount of financial demand. If one considers the usually small numbers of "products" the principle investigator will need to address small clinical trials, these high risk investments will not easily find the internal support needed. Nevertheless, in the long-term, once implemented into a working system, this newly implemented system will lead to less hassles and burdens to carry out additional cell therapy studies and significantly promote the standardisation in

**4. Standardizing new cell therapies for PD: The TRANSEURO project**

As discussed in the introduction of this chapter, in recent decades several clinical studies using cell therapy in PD were investigated often producing large differences in the outcome (Winkler, Kirik & Björklund 2005h). In many cases it was found that the protocols needed to conduct the therapy varied significantly in many places and a generalized statement about the efficiency of the studies was difficult to reach. In parallel, the legislation has only

actually technically difficult to prove.

to finish the validation activities.

multicenter trials.

Cell transplantation for PD-patients is a promising therapy, based on the previous preclinical and clinical research. The use of foetal tissue as proposed in the TRANSEURO project holds a number of (ethical) concerns and technical handicaps, but is an ideal candidate to merge basic science and industrial standards to achieve an efficient therapy. This may set the basis of future cell therapeutically approaches with considerably more complex processes and thereby noticeable higher regulatory demands. The worldwide effort working with embryonic stem cells and with induced pluripotent stem cells (iPS) might in the near future generate alternative candidate cells for clinical therapy in PD. However, complicated manufacturing processes involving gene manipulation, advanced cell culture techniques and risk of tumorigenicity will benefit greatly from the currently planned

Transplantation of Foetal Ventral Mesencephalic Grafts

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#### **6. Acknowledgement**

The authors are partially funded by the European Commission under the 7th Framework Programme – HEALTH – Collaborative Project Transeuro (Contract n°242003) – www.transeuro.org.uk

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brain for function: use in a model of Parkinson's disease." *Research Publications -*

### *Edited by David I. Finkelstein*

Parkinson's disease (PD) is characterised clinically by various non-motor and progressive motor symptoms, pathologically by loss of dopamine producing cells and intraneuronal cytoplasmic inclusions composed primarily of ?-synuclein. By the time a patient first presents with symptoms of Parkinson's disease at the clinic, a significant proportion of the cells in the substantia nigra have already been destroyed. This degeneration progresses despite the current therapies until the cell loss is so great that the quality of normal life is compromised. The dopamine precursor levodopa is the most valuable drug currently available for the treatment of PD. However for most PD patients, the optimal clinical benefit from levodopa decreases around five to six years of treatment. The aim of the chapters of this book is to work towards an understanding in the mechanisms of degeneration and to develop disease modifying therapies.

Towards New Therapies for Parkinson's Disease

Towards New Therapies for

Parkinson's Disease

*Edited by David I. Finkelstein*


*3.1.4 Synthetic methods in preparing 2,4,5,6-Tera-substituted pyrimidines*

One main strategic approach employs the step-wise replacement of leaving groups that exist on the already constructed pyrimidine (like 2,4,6-trichloropyrimidine (109)). The selection of the substituted pyrimidine as starting material depends on the

**Figure 23.**

*Synthesis of* N*-2,4,6-trisubstituted pyrimidine derivatives as potent aurora a kinase inhibitor. Reagents and conditions: a) Et3N, EtOH, 0°C; b) TsOH.H2O, n-BuOH or 1,4-dioxane, 100–140°C; c) 1,4-dioxane, under microwave, 150–180°C.*

desired product i.e. in case of 2,4-disbustitited pyrimidine the synthesis starts with 2,4-dihalopyrimidine and in case of 2,4,6-trisubstitutedpyrimidine the 2,4,6-trichloropyrimidine (109) is picked. Additionally, the reaction conditions and reagents used in the chemical process is by virtue reliant on the type of chemical bond formed or the type reaction employed. The order of applying the sequential displacement of the leaving groups is contingent on the type and properties of the functional group and conditions that ensure better purity and higher yield of the intermediates, building blocks or the final products. A special attention will be devoted to recent reports related to synthesis of FDA approved drugs or in few cases such derivatives with high potential bio-activities.

Regioselectivity is mostly guaranteed by picking the proper starting material and the fitting sequence of substitution (**Figure 24**).

In a recent study Zhang and colleagues reported the synthesis of anisole containing 2,4,5,6-tetrasubstituted pyrimidines [92]. The team exploited the commercially available reagents like properly substituted malonic acid diesters (124) and guanidine hydrochloride (125) that were condensated in anhydrous methanol under slightly

**Figure 24.**

*Examples of 2,4,5,6-tetrasubstituted pyrimidines. R = H, methyl-, ethyl-, propyl-, isopropyl-, Propargyl-., R = allyl-, butyl-,* sec*-butyl-, phenyl-, benzyl-, Fluoro-.*

### *Synthetic Approaches for Pharmacologically Active Decorated Six-Membered Diazines DOI: http://dx.doi.org/10.5772/intechopen.109103*

basic conditions (sodium methoxide) to produce the 5-substituted 4,6-dihydroxy-2 aminopyrimidine intermediate (126). (126) was converted to the 5-substituted 4,6 dichloro-2-aminopyrimidine derivative (127) upon treatment with Vilsmeier–Haack– Arnold (VHA) reagent [93]. Cations should be taken prior to treating 5-substituted 4,6-dichloro-2-aminopyrimidine derivatives with (chloromethylene) dimethyliminium chloride (VHA, 130) (**Figure 26**).

Using VHA reagent, drying of the starting materials and conducting the reaction under inert conditions helped in affording the final products in higher yields and purity compared to the previously reported reaction conditions (such as chloride donating mineral acids such as POCl3, PCl5, SOCl2, or COCl2 with diverse additives like DMF, pyridine, 2-methylpyridine, diphenylamine, or triethylamine) reported to end in less than 30% yields and complicated purification procedures [94]. Following the deprotection of the 2-(dimethylamino) methylene protecting group using hydrochloric acid the desired 4,6-dichloro-5-substituted-2-aminopyrimidines were isolated and purified. The 4,6-dichlorides were sequentially displaced under nucleophilic substitution conditions. The first 4-chloro was substituted by aniline derivatives in refluxing ethanol (see step c in **Figure 25**) while the second chloride was exchanged for anisole (4-methoxythiophenol) employing sodium tert-butoxide (1.33 mmol), under heating at 82°C (see step d in **Figure 25**, [92].