Developing a new drug from the laboratory into the medical formulary to improve patient care involves many experts from multiple disciplines. From the initial concept of a new drug with a demonstrable effect in cell culture or a new diagnostic system with higher sensitivity, a development team and a business team must be integrated to carry out the complicated drug development process. Successful product introduction can take ten years or longer without the right expertise. Alternatively, with planning, the right team and a little luck, the concept can be moved to market in as little as 3 to 5 years. Since we are in the pharmaceutical industry, our initial focus is on the technical processes with the initial idea, followed up with a preclinical prototype that is tested in animals for efficacy and safety. A formal clinical prototype with supporting regulatory documentation is then tested in Phase I clinicals for safety. Then a final prototype with more extensive safety and stability testing undergoes process development for manufacture of clinical supplies for Phase II dose ranging clinical studies and Phase III pivotal efficacy studies involving hundreds of patients. In parallel, continued stability testing, completion of analytical protocols and manufacturing scale-up are carried out. These activities come together in a new drug application (NDA) for regulatory approval. While the NDA is reviewed by regulatory authorities, the manufacturing facility is completed and the process validated. Analytical methods are transferred to the quality control organization and analysts, equipment operators and support personnel are trained in the standard operating procedures that have been defined to manufacture the final product. If all goes well, the final step in regulatory review is the pre-approval inspection of the manufacturing facility, followed by approval and product sales to patients needing the new regimen. In these final steps before product launch, the business and regulatory team will have secured formulary approval, initiated an educational campaign to make the doctor aware of the new treatment, and set up a distribution system to get the drug to the patient. Now stop and think. No single person has the expertise to direct all of the processes alone. Over the next few days, we will explore what skills are needed and how to plan for success in integrating the right people into getting a new drug concept into the market.

The in-licensing of new products and technologies plays a critical part in the portfolio of products for Merck & Co. Several examples will be given of in-licensed products that have been successfully developed and marketed worldwide. Merck & Co. continues to be very active in pursuing opportunities for new products through external sources. The process by which Merck & Co identifies, evaluates and negotiates potential sources of external innovation will be outlined. The current structure of the groups with primary responsibility for licensing activities will also be described. Finally, the advantages of partnering with a company such as Merck & Co will be described.

It is little surprise that university professors are the most likely founders of new biotechnology companies. As holder of advanced degrees, often with years of post-doctoral training experience, professors are among the most highly educated professionals. Successful independent principal investigators in universities must be able to conceive, undertake and report the findings from new research initiatives; recruit, train and manage personnel; raise funds, budget and perform financial accounting; and be effective public speakers. These are all the ingredients that the high tech industry looks to find in its leaders. Finally, the high technology arena demands a robust level of creativity and inventiveness for a new company to effectively compete. Small wonder then that most companies spin out of universities that provide facilities to incubate discovery and innovation. Sophisticated universities have evolved effective industrial liaison offices that facilitate the birth of new companies. They can provide the founding scientists with useful advice, particularly with respect to the patenting and licencing of intellectual property (I.P.). As much of the early conception and demonstration of utility of I.P. tends to arise from activities carried out in the university, the host university is a co-owner of the I.P. with the founding scientists. It is critical that an effective, open partnership between the two is established early on as the scientists could be treading dangerously close to conflict of interest quagmires. Businesses require a much greater level of funding to operate than the typical university laboratory, so it is necessary for the founding scientists to also align themselves with experienced business expertise. This often also requires alliances with venture capitalists and high net worth investors. For those scientists that are not fixated on making money as their prime motivation in life this can be a problematic relationship for the scientists and business men alike. In the struggle for ultimate control of a fledgling company, it is typical for scientific founders of companies to eventually return to their academic research laboratories and leave the running of their companies to the "experts." The scientists may simply tire of trying to meet both their industrial and their academic research, teaching and administration obligations. They may even face scorn from academic colleagues that question their commitment to academic affairs, and their business colleagues that demand full attention. A common outcome of this scenario is that the resulting companies often loose their defining vision with the loss of their scientific founders, and not too surprisingly develop into unexciting, licencing opportunity-type companies with limited growth potential. Great companies tend to have unique platform technologies that offer tremendous opportunities to the market place. The founding scientists have the expertise to foster the development of such revolutionary technologies, and remain vital to the long term success of these companies. For a high tech company, strong science remains a critical element in its bottom line financial success. spelech@home.com

In the past decade many Canadian universities have made a major commitment to increasing their collaboration and partnership with industry for effective commercialization of university-based research and innovation. Universities are now the major engines for creating a knowledge-based industry and economy in many Canadian cities. These universities have built technology transfer offices (TTO) that are staffed with highly experienced people, with both scientific and business backgrounds. The staff of TTOs are capable of assessing the commercial potential of innovations, protecting the intellectual property, marketing and licensing. In addition to their licensing activities, many universities have taken on the challenge of creating start-up companies based on platform technologies arising from university laboratories. For this purpose, many TTOs also collaborate with a number of venture capital groups specialized in investing in early stage technologies. Governments and research funding agencies strongly support technology commercialization activities at Canadian universities. In order to have successful technology transfer from universities, it is essential that TTOs have support of researchers, governments and the business community.

1 Effective university technology transfer is only possible if all parties concerned understand and realize the value of this activity and work in partnership.

2 A TTO must realize its mandate is to capture the maximum commercial value of curiosity-driven research, but not to convert universities into commercial research centers.

3 To increase the commercial value of research results, funds must be provided to protect and strengthen the intellectual property position.

4 University administration must be flexible in dealing with numerous issues and problems associated with the commercial activity of their faculty members/employees.

5 A TTO must serve its clients (i.e., researchers) well in order to gain confidence and become an effective agent for university researchers.

An increasing number of faculty researchers are doffing their lab coats at the end of each day and donning their business suits as they embark on their evening career as businesspersons. Has the professor by day now become an entrepreneur by nights? As a new company develops from an initial concept, through incorporation, start-up and virtual stages, to a fully operational company, what are the changing roles of the scientist? Researchers now find themselves in new roles as shareholders, directors, CEO’s, founders and more - all while trying to maintain an active lab on campus. How do you maximize the benefits of the university support for the start-up, government leverage of company research, while managing the conflict-of-interest issues which have now emerged?

A key piece in the drug development process is the generation and efficient use of high quality preclinical safety data. Development goals vary widely between companies. For the vast majority of start up companies, the objective of development is not to take the candidate drug through the entire development process alone, as the costs (and risks) are overwhelming. Rather, the objective may be more limited, such as the generation of proof-of-concept data in patients in order to attract funding partners, and therefore, companies need to enter into clinical trials as rapidly and efficiently as possible. In North America, this means the preparation and filing of an Investigational New Drug Submission/Application (IND). In the regulatory arena, small start ups and large multinational companies alike are held to the same high standards, so therefore, a well conceived nonclinical management strategy is essential to offset some of the research and development costs that consume precious capital, avoid regulatory delays, and allow for rapid movement into the clinic. The preclinical data include the results of animal toxicology studies, toxicokinetics, in vitro and in vivo mutagenicity/genotoxicity studies, safety pharmacology, and pharmacokinetic data, which compliment and aid in the interpretation of the safety data. These data are necessary to understand the compound under development, but their generation is not solely a scientific exercise, as the results must be provided to, and reviewed by, regulators prior to initiating clinical trials. At a minimum, the pivotal safety studies must be conducted in accordance with Good Laboratory Practice (GLP) regulations. In the case of the animal toxicology studies, the results should identify the target organs for the new drug, the dose-response relationship, reversibility of effects, and aid in the selection of doses for the initial (first-time-in-man) Phase I clinical trial(s). The design of the studies themselves should be tailored specifically for the drug under development, utilizing information obtained from published and unpublished sources for related agents, drug classes, and agency expectations (sources of such information will be discussed). Ongoing efforts related to the international harmonization of the requirements for new drug development have removed many of the jurisdictional differences in requirements for preclinical testing, such that a typical toxicology program to support a Phase I IND for a new chemical entity (NCE) includes the minimum following studies: 28-day rodent toxicology study (usually the rat); 14-day nonrodent toxicology study (usually the dog); in vitro genetic toxicology studies (bacterial mutagenicity and mammalian chromosomal aberration); and safety pharmacology (i.e. cardiovascular safety), depending on the nature of the agent, the route of administration, etc. Other data required would include the results of nonclinical efficacy studies, and some early nonclinical pharmacokinetic data, and of course, data demonstrating the quality and stability of the agent/formulations used in the nonclinical safety studies and proposed for the clinic. It should be noted that preclinical development programs can vary greatly depending on the indication (i.e., oncology versus hypertension), duration of treatment (i.e., single dose versus chronic treatment), and nature of the agent under development (i.e., small molecule versus protein). The timing and the cost of studies to support clinical trials and eventual registration will be discussed. Start up companies can have limited in-house resources, such as finances, personnel, and a limited pipeline, and must by necessity contract out toxicology studies to contract laboratories. In the case of Canadian companies, effective use of resources in Canada can result in significant tax credits. Effectively dealing with these laboratories (protocol development, bid solicitation, study placement, GLP monitoring, and draft report review) is essential for the generation of good data and for a successful safety testing program, but it must all begin with a well-designed testing strategy and end with a high quality regulatory filing.

Assuming human drug elimination is linear and completely mediated via hepatic P450 oxidative enzymes, in vivo (unbound) intrinsic clearance may be estimated in vitro from human cadaver microsomal preparations. This value may be used in concert with other in vitro determinable variables to estimate in vivo human drug clearance. Allometric methods may also be used to predict human pharmacokinetic parameters based on parameters from laboratory animal studies. In particular, human clearance may be predicted within a 2-fold error (100% above or 50% below) of the observed value with a probability of about 60-70%.

Two principal factors control oral drug delivery, drug solubility in the gastrointestinal tract and intestinal membrane permeability. Systemic availability can be further limited by first-pass metabolism. Recent advances in tissue culture methodologies and correlation methods have allowed the establishment of rough guidelines for selection of drug candidates based on intestinal membrane permeabilities. Candidates for oral controlled release can be selected based on their intestinal membrane permeability. Drug solubility is also screened, today, in the major pharmaceutical companies. While solubility is the principal focus, that is solubility in water, it is really solubility in the gastrointestinal tract and, in particular, the duodunum and jejunum that are the critical factors. Solubilization is really the most important criteria. Solubility both effects the maximum potential driving force for mass transport across the intestinal membrane as well as the drug dissolution rate. Solubility itself is not as critical a parameter as the dose to solubility ratio or dose number. Very low solubilities are acceptable for very potent low dose compounds, assuming that in vivo solubilization occurs. Finally, first-pass metabolism can limit systemic availability and this is often true for insoluble drugs which are hydrophobic. Correlations between in vitro clearance and in vivo clearance are increasingly utilized today to select drug candidates which minimize first-pass metabolism. Three factors, solubility, permeability and first-pass metabolism can be roughly screened for today. However, it is my general impression that the selection for controlled release delivery is not a consideration in the pharmaceutical industry at this time. This is unfortunate because oral controlled release offers an approach to optimizing pharmacodynamics via the oral route. With an increasing interest of regulatory authorities in pharmacodynamics and surrogates for therapeutic response, the opportunity for optimizing oral drug delivery is present.

With changes in the medical field, major changes have occurred in pharmaceutical industry during the past decades. Time to market has become crucial and that demands an expedited drug development process. Not only each development phase has to be efficient, the transfer processes linking various stages are also critical. A smooth transition from product formulation to clinical supplies has been a major undertake for formulation scientist and engineers. From bench scale preparation, the formulations need to be scaled up to Phase I, II and III clinical supplies efficiently. Formulation scientists need to consider scale up at the early onset of the project. Material needs to be selected, proper documentations are required, formulation/process parameters have to be defined. Packaging and labeling requirements of the clinical supplies also need to be considered. This presentation will discuss the importance of each step in bringing a formulation from laboratory to clinical supplies. Specific examples encountered will be presented as well.

The development and commercialization of a pharmaceutical product requires extensive analytical support. Effective management of both internal and external analytical resources is critical to meeting drug development timelines. Collaboration between analytical and other scientific disciplines, quality and regulatory functions are essential ingredients to a successful drug development program. Analytical Chemistry begins with the characterization of the synthesis and/or purification for the active pharmaceutical ingredient (API). Analytical Development continues as the API is formulated into a drug product for pre-clinical and then IND submissions for human clinical evaluation up through NDA submissions for approvals to market. In the pharmaceutical industry the Quality Control (QC) function typically becomes involved as the API and drug product require elements of Good Manufacturing Practices (cGMP’s) including testing and release of each lot for human clinical use. Depending on a pharmaceutical organization’s functional structure the transfer of responsibility from an Analytical R & D function to a QC function could be at the IND stage or up to NDA approval. The API will require physical-chemical characterization that leads to the establishment of specifications including initial estimates of stability and storage requirements. The safety (carcinogenicity, etc.) of the API and major impurities will need to be established. The route(s) of administration will dictate analytical and microbiological method development, testing, and specification requirements in support of formulation development. Many companies have had difficulties due to problems in the basic manufacturability of the product, scalability, and batch-to-batch reproducibility. Are there any unusual excipents? Knowing the purpose of each ingredient will aid in defining tests and specifications that will control desired properties. During process development, careful attention to the development of robust in-process control tests is often critical to producing consistent product. Are there novel processing steps or equipment that may require the development of specific tests? Effective process characterization will determine what steps are critical to control, and can affect the stability of the product. The development of methods to assess equipment cleaning procedures is critical for multi-purpose facilities and equipment. Both analytical chemistry and quality control may support container closure system suitability testing and for sterile products, sterile process validation. Process validation support, and fully developed quality systems, including instrument qualification, within the QC lab will be requirements for a successful pre-approval inspection. When transferring methods from Analytical R & D into QC address the following questions. What is the status of method development and validation? What is the training and experience of the personnel in both labs? Are there facility and equipment differences between the two laboratories? How complex, characterized, and well developed is the product formulation and manufacturing process? Jointly approve a lab-to-lab method transfer protocol. What is an acceptable difference between labs given the intended purpose of the test, the specifications, and known accuracy and precision of the test method? If these and related questions are addressed the transition from analytical chemistry to routine quality control can be a competitive advantage in accelerating the commercialization of new drug products.

To be successful, companies must be able to bring products from the laboratory to production to market quickly and efficiently. This usually means that the product must be scaled up and launched in a short amount of time in order to quickly capture market share. The product must also be manufactured in high volume and at low cost. In the pharmaceutical industry, first to market is a very important factor to establish market share since gaining market share later with a second or third to the market product can be very difficult. Process development takes products developed in the laboratory and transfers them to manufacturing. This usually involves scaling up the product to make it in sufficient quantity for manufacturing. Significant hurdles occur at this stage where something that is simple in small scale becomes extremely cumbersome in larger scale. Establishing what constitutes a full size lot is a balancing act. Larger lot sizes means more difficult handling, more reconciliation issues, shelf life issues and larger (more costly) qualification runs but can also result in fewer lots to test, more efficient production flow, etc. Validation runs would normally require three lots with at least one lot full batch size and two partial batches at a minimum. If there are significant issues with larger or longer processing runs, three full size runs would be more appropriate. If there are various product configurations, additional lots would have to be produced. One area that is frequently neglected due to time constraints is product characterization. Understanding the product and what factors can affect product performance is vital to gaining and keeping market share as well as increasing profitability. Raw material vendors can change or discontinue the product, process conditions can vary outside production limits, environmental controls not maintained, equipment failure, etc. any of these can seriously impact product performance and potentially shut down manufacturing. Product characterization is building quality into the product and can save time and money over the life of the product and is especially vital to keeping manufacturing producing product. Validation requires that the manufacturer has knowledge about the product and knows how to make it consistently. That is where product characterization is used to establish which parameters will or will not be evaluated. Experimental design can be and should be used to help identify critical process parameters. It is highly recommended to take the time and effort to understand the product. It will result in fewer questions of product quality if difficulties arise during the manufacturing of the product. Scale-up can be a difficult process since it requires a great deal of work in a short amount of time. The benefit is that you will learn the most about the product during this phase. Don’t underestimate the amount of time, money and resources that will be required, it will always take more than you anticipated. In the end, however, it will pay dividends for years to come.

National Agencies that are responsible for regulating therapeutic products are mandated to ensure that drugs, medical devices and like articles available in their jurisdictions are safe, effective and of high quality, and that there are sufficient and proper information and controls available regarding their benefits and risks, and accessibility. These authorities are meant to be in the best interests of consumers primarily, and also of researchers and manufacturers. In Canada, regulatory authority is wielded by the Therapeutic Products Programme of Health Canada; in the USA, the FDA’s Centres for Drug and Biologics Evaluation and Research (CDER and CBER) have similar authorities. The two countries have similar requirements and follow similar processes regarding manufacturers’ Investigational New Drug Submissions (INDs) and New Drug Submissions (NDSs, Canada) and Applications (NDAs, USA). There is a coalescing of the technical regulatory requirements in North America, Europe, and Japan through the ICH process, which is expected to have a continuously greater impact on the introduction of new products. The mechanics of the regulatory processes in Canada will be described. Some differences between FDA’s and TPP’s approaches will be discussed. Observations on best practices on the part of companies and regulators that enable decisions to be reached in a timely, efficient and fair manner will be presented. Newly-developing areas that are attracting the attention of manufacturers, the public, health care providers, and regulators, and the challenges posed to all by the easy accessibility of a glut of information and misinformation will be briefly discussed.

Phase I Clinical Trials: Outlook From Canadian CRO Perspective
M. LeBel, Anapharm Inc., Ste. Foy, Québec

In the pharmaceutical world, time spent on clinical studies is money. For example, for a drug with a potential of 365 Million dollars of sale a year, each day of delay in clinical development is worth 1 million dollars. With the upcoming change in regulation at HPB, a notification system with a target review time of 48 hours is proposed for Phase I studies. This may compare advantageously with the zero day waiting in Holland, UK and Germany and the 30 day in USA. However the time to prepare a submission to HPB will equal or exceed the time to fulfill requirements for submission in these countries. Canadian CRO like Anapharm may cut short the time before a Phase I study start by using an efficient IRB that can meet every week and provide a written answer in 72 hours. The one week required by IRB at Anapharm compares favorably to two to three weeks in Holland, UK and France. The generation of final report in CRO should be similar with some French CRO claiming to perform this task in 2 weeks.

Maintaining Balance: Research and Commercialization
G.D. Lopaschuk, University of Alberta, Edmonton, Alberta, Canada; L. Humphreys, Alberta Heritage Foundation for Medical Research, Edmonton, Alberta, Canada

Research advances by academic researchers that have commercial potential are relatively abundant in Canadian Universities. What is less common is having academic researchers with the necessary skills to commercialize these research advances. As an academic researcher at the University of Alberta, Gary Lopaschuk found himself in just such a situation. His research in the area of energy metabolism in the heart resulted in the development of a number of technologies that had commercial potential. However, his background training had ill prepared him to even begin to commercialize this technology. It was also necessary for him to balance the time necessary to commercialize this technology with his commitments to the University. In the first part of this presentation, Gary Lopaschuk will discuss the hurdles an academic scientist must overcome in commercializing a research idea. He will discuss the important issues that needed to be addressed, including: 1) initially forming his company, Metabolic Modulators Research Ltd. (MMRL), 2) how MMRL would interact with the University, recognizing that every technology requires a custom arrangement, 3) intellectual property issues, 4) how to operate a University spin-off company within a University environment, 5) developing a business plan, and 6) raising funds to move the research technology towards the market place. During this process, the availability of resources and technology commercialization expertise was critical. For MMRL this included the expertise and resources of the University of Alberta Industry Liaison Office, the National Research Council Industrial Research Assistance Program, and the Alberta Heritage Foundation for Medical Research (AHFMR). The second part of this presentation will describe the role of the AHFMR in this process. The AHFMR mission is to support basic, health and medical research that leads to improved health and health infrastructure. Gary Lopaschuk has been supported by the Foundation as a Scholar and Scientist for over 13 years. Total funding, including establishment grants and trainee awards related to Gary’s laboratory have exceeded 2 million dollars. AHFMR also has a Technology Commercialization (TC) Program that is intended to meet the very early needs of researchers embarking on the innovation highway. Gary’s company, MMRL, has recently received funding from this program to advance the commercial development of his technology. Supporting Gary Lopaschuk and his teams in both the science and commercialization process, Linda Humphreys will discuss the competing and complimentary issues from the AHFMR perspective. The focus will be on the role of the TC Program in relation to other sources of funds and services, and how these efforts compliment the objectives of the researcher, the institution and the mandate of AHFMR.

Purpose:This randomised double-blind study examines the efficacy and safety of lignocaine and bupivacaine following administration of lignocaine alone and in combination with bupivacaine to the fallopian tubes of 51 women undergoing tubal ligation. The main limitation of lignocaine is its relatively short absorption half life compared with bupivacaine.  In this study it was hypothesised that a combination of the two local anaesthetics, bupivacaine and lignocaine, injected into the fallopian tube prior to fitting of the Filshie® clip would give a longer duration of analgesia than lignocaine alone. Recognising that a rapid onset of action is still needed for pre-emptive analgesia, a combination of anaesthetics is  preferred to bupivacaine alone.

Method: Each patient was randomly assigned to either Group A, administered  lignocaine 2 mL of 1 % injection on each side (total dose,40 mg) or Group B, administered a combination of 1 mL of 2 % lignocaine and 1 mL of 0.5% bupivacaine on each side of the two tubes (total dose, 40 mg lignocaine + 10 mg bupivacaine). Blood samples (6 mL) were taken from the right antecubital fossa immediately before administration of the local anaesthetic and at specific times upto 180 min after the administration of local anaesthetic(s) into the fallopian tubes. Patients were administered pain intensity questionnaires which consisted of a visual analogue scale (VAS) pain score and a modified McGill questionnaires.

Conclusion: The purpose of this study was to investigate the rate of systemic removal of lignocaine and bupivacaine from the fallopian tubes in women undergoing tubal ligation. It was expected that lignocaine would have a fast onset of action whilst bupivacaine would provide more sustained blood levels resulting in a longer duration of anaestheic cover. However, the results of this study show that systemic uptake of both lignocaine and bupivacaine from the fallopian tubes is too rapid for any real difference in efficacy to be observed. Thus the rapid and non-stereoselective absorption of bupivacaine from the site of administration in tubal ligation does not support the addition of either rac-bupivacaine or S(-)-bupivacaine to lignocaine for this indication.

Purpose. To study the fetal/maternal exposure of the enantiomers of fluoxetine (Fx) and its N-demethylated metabolite, norfluoxetine (nFx), in the pregnant ewe and fetus after chronic maternal intravenous infusion.

Methods. Three pregnant sheep were surgically prepared for pharmacokinetics studies at late gestation (115-120 d, term 145 d). A 70 mg bolus iv injection was given to the mother followed by an 8 day continuous infusion of racemic fluoxetine at a rate of 6.92 mg/h to achieve steady state concentrations (Css). Serial maternal and fetal blood samples were collected at 0, 5, 15, 30 min, 1, 2, 4, 6, 12 and 24 h for the first day, followed by 12 h sampling thereafter. The infusion was terminated on day 8 and post-infusion blood samples were collected at 0, 5, 10, 20, 30, 45 min, and at 1, 2, 3, 4, 6, 9, 12, 24, 36, 48, 60, and 72 h. Plasma concentrations of Fx and nFx enantiomers were determined using GC/MS.

Results. Both Fx and nFx were extensively transferred across the placenta with a fetal/maternal Css ratio of 0.75 and 0.51, respectively. The fetal plasma terminal elimination half-life of Fx (71.6 h, n=2) and nFx (808.8 h, n=2) was considerably longer when compared to the maternal values (29.3 h for Fx (n=2), 56.5 ± 20.4 h for nFx). In addition, stereoselective disposition was observed for the Fx enantiomers with an S/R Css ratio of 3.52 and 3.18 in mother and the fetus, respectively. Steady-state maternal total body clearance for S-Fx and R-Fx was 0.50 and 1.92 L/h/kg, respectively. In contrast, stereoselectivity was not observed for nFx with S/R Css ratios nearing unity.

Conclusions. Fluoxetine and its N-demethylated metabolite, norfluoxetine, exhibit extensive placental transfer in sheep. Both Fx and nFx exhibited a prolonged elimination half-life in the fetus when compared to the adult. The differences between the maternal and fetal steady state Fx and nFx S/R ratios suggest that stereoselective disposition of Fx may result from another elimination processes other than N-demethylation.

This study was funded by the Medical Research Council of Canada. Caly Chien was a recipient of University of British Columbia Graduate Fellowship.

Presented at American Association of Pharmaceutical Scientists Annual Meeting, Nov 14-18, 99 at New Orleans, LA. Published in: AAPS PharmSci. 1(4): s224, 1999.

Purpose: The purpose of this program was to design novel, non-steroidal high affinity ligands for the estrogen receptor.

Methods: A ligand-based de novo design approach called Evolutionary Molecular Design™ (EMD) (U.S. Patent # 5,699,268), proprietary to Nanodesign® Inc. of Guelph, Ontario, Canada, was used to design novel, non-steroidal high affinity ligands for the estrogen receptor. EMD uses information about the structure and biological activity of known pharmacologically active compounds ad criteria to evolve new structures that share and improve upon the properties of the original compounds.

Representative compounds from the EMD process were synthesized and tested in three in vitro assays, MCF-7 Whole Cell Antiestrogenicity Assay, Estradiol Competitive Binding Assay, and the Gene Transfection Assay.

Conclusions:

1. EMD generated novel compounds that were predicted to have high affinity for the estrogen receptor.

2. Subsequent synthesis and biological testing of these compounds confirmed the prediction by EMD.

3. EMD is a powerful ligand-based de novo design platform technology which can enhance and accelerate pharmaceutical discovery efforts.

Supported by MRC 983587.

Introduction: Ibuprofen is a chiral non-steroidal anti-inflammatory drug, available as the racemate. In rats and humans, the main clearance pathway of R-ibuprofen is its chiral inversion to S-ibuprofen. In patients with dental surgery pain, the stereoselective disposition of ibuprofen in plasma is reversed, hence the concentration of the active S enantiomer is lower than that of the R enantiomer due, perhaps, to a cytokine-induced suppression of the inversion pathway. In addition, pro-inflammatory cytokines suppress the metabolism of many drugs. As ibuprofen is also indicated for the treatment of inflammatory conditions, such a disease-drug interaction may be of clinical importance.

Purpose: The objective of the study was to compare differences in the body distribution of 14C labeled Azidothymidine (AZT) bound to a colloidal drug carrier system with a control solution.

Methods: Polyhexylcyanoacrylate nanoparticles were prepared by emulsion polymerization in the presence of AZT and an ionic emulsifier bis (2-ethylhexyl) sulfosuccinate sodium. The AZT-control solution was equally prepared, but without the monomer. The two preparations were administered either by i.v. injection or orally. After determined time points the animals were scarified. The cadavers were shock-frozen in cellulose gel and cut into slices using a cryo-microtome. The tissue cross sections were fixed on an adhesive tape and were freeze-dried. The quantification of the radioactive AZT in the different organs and tissues was performed by radioluminography, and the images were generated on a computer.

Results: After i.v. injection of AZT-nanoparticles, a high drug concentration was found in the organs belonging to the reticuloendothelial system. In these organs the radioactivity was inhomogeneously distributed showing that the uptake of the particle-associated radioactivity was depended on the type of the cells located in the organ and was consistent with uptake by macrophages. The highest radioactivities were found in the gastrointestinal-tract and in the liver. A difference in the elimination pathway between AZT-control solution and AZT bound to nanoparticles also was visible on the images. Similar results were obtained after oral administration. However, with the latter route a larger portion of AZT remained in the GI-tract especially after administration of nanoparticle-bound drug.

Conclusion: The results demonstrated that radioluminography is a useful method to visualize the macrophage targeting and change in body distribution of 14C labeled AZT bound to nanoparticles.

Purpose: The dissolution behavior of high permeability/high solubility drugs (class I, Biopharmaceutical Drug Classification System, BCS) and high permeability/low solubility drugs (class II) was tested in various media. The aim of the study was to investigate whether the use of biorelevant dissolution media (BDM) would be advantageous over the use of standard media for predicting the in vivo performance of these drugs.

Methods: The dissolution tests were performed using USP 23 apparatus 2. Conventional buffers and USP media were compared with two BDM containing different amounts of lecithin and sodium taurocholate.

Results: The dissolution of metoprolol (class I drug) was rarely influenced by the nature of the dissolution media. For class II drugs such as glibenclamide, the dissolution behavior showed differences in all media tested. The dissolution results of the two glibenclamide formulations were compared with those from an in vivo bioequivalence study undertaken by the central quality control laboratory of the German pharmacists (CL). The bioequivalence criterion set by the CL requires more than 80 % drug release within 10 minutes. Results in fasted state simulated intestinal fluid (FaSSIF), one of the BDMs, met the CL criterion and this medium was also able to discriminate between the two formulations. This was not the case for any other media tested. An improved in vitro/in vivo correlation (IVIVC) could be shown between the dissolution results of the tested formulations using FaSSIF and the in vivo bioequivalence study.

Conclusions: The study indicates that BDM are better able to discriminate between glibenclamide formulations than standard dissolution media.

Purpose: Solutions of the amino acid glycine are used for organ irrigation during abdominal and musculoskeletal surgeries, and significant systemic absorption of glycine during these procedures has been reported. In addition, glycine is used as an inactive ingredient in intravenous drug products and as a component of total parenteral nutrition amino acid injections. Post-surgical adverse events have included local and cerebral edema, visual effects (including blurring or loss of acuity) and immune-mediated symptoms. To more fully understand the potential risks, the safety of intravenous glycine has been evaluated.

Methods: Critical evaluation of all non-clinical and clinical literature pertaining to intravenous glycine.

Results: In clinical investigations conducted with healthy male volunteers, reversible visual effects (acuity and visual evoked potentials) were observed in one subject following a bolus intravenous dose of 4.4 g glycine in 200 ml over 5 minutes. Similar effects were also observed at doses of 15 to 22 g, infused over a period of 20 minutes. The absence of a dose- or time-dependent relationship, and the mild, transient nature of the visual effects suggest that bolus intravenous doses of up to 22 g of glycine are well-tolerated. Animal data for intravenous glycine administration support the reported effects of systemic glycine exposure in humans. Transient effects on visual parameters (diminished visual evoked potentials) were observed in dogs following single doses of 1 g/kg, while single doses of glycine in sheep equivalent to 0.9 g/kg produced reversible behavioural blindness and loss of pupillary response.

Conclusion: Based on these data, it is concluded that serious adverse events are not likely to occur in response to a bolus intravenous dose of up to 22 g glycine (i.e., 0.44 g/kg in a 50 kg adult).

Mometasone furoate (MF), is a potent synthetic glucocorticoid, shown to have an increased ratio of local to systemic activity in the treatment of inflammatory disease. It is marketed as a topical formation (Elocon), nasal spray (Nasonex) and a dry powder aerosol (Asmanex).

Purpose: The objective of this study is to compare and characterize the kinetics of the in vitro metabolism of MF, using a rat model as well as human tissues.

Methods: The in vitro metabolism of MF was evaluated in 9000g supernatants of tissues with protein content of 4 mg/ml including human lung, rat lung and liver, intestine, and stomach, as well as rat and human plasma. The MF remaining and metabolites formed were measured by HPLC using a Beckman ultrasphere octyl column and a mobile phase of 60% methanol and 40% water, with UV monitoring at 248 nm.

Results: In Sprague-Dawley rats, MF was predominantly metabolized by liver, followed by plasma and lung tissues, but negligibly by intestine, and stomach. A metabolite (M4), chromatographically more polar than MF, was formed rapidly in rat liver, minimally in intestine, stomach and lung, with an NADPH-generating system. In contrast, small quantities of three other metabolites (M1, M2, M3) were observed in rat and human plasma, and in rat liver, intestine, stomach, lung tissues and human lung tissue without the NADPH-generating system. In vitro metabolism of MF was qualitatively similar in the lung and plasma for humans and rats. A similar in vitro metabolism profile of MF was observed for rat and human lung, and between rat and human plasma. The degradation of MF was faster in rat plasma (t0.5 = 7.5 ± 2.1 h) than in human plasma (t0.5 = 18.3 ± 4.3h).

Conclusion: The rat appears to be a suitable model for metabolism of MF as it qualitatively responds in a similar fashion to humans. Further, studies are ongoing to investigate the predictive value of this model of MF metabolism in other human tissues.

*Mometasone furoate was kindly supplied by Schering-Plough Australia Pty Ltd.

Cyclohexyladenosine (CHA) is an adenosine A1 agonist and D,L-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849) is a competitive N-methyl-D-aspartic acid (NMDA) antagonist. Their influence upon the anticonvulsant activity of intraperitoneally administered sodium valproate, diazepam, diphenylhydantoin, lamotrigine, phenobarbital and carbamazepine was investigated in mice. Convulsive seizures were induced by the use of electroshocks and pentylenetetrazole (PTZ). CHA (7 mg/kg,i.p.) and CGP 37849 (10 mg/kg, i.p.) were found to enhance the anticonvulsant activity of the tested antiepileptic drugs against both electroconvulsions and PTZ-induced convulsions. Both CHA and CGP 37849 significantly decreased the ED50 values of these drugs against both electroconvulsions and PTZ-induced convulsions. CHA (7 mg/kg, i.p.) and CGP 37849 (10 mg/kg, i.p.), alone or in combination with the tested antiepileptic drugs produced no significant effects on the heart, body temperature, behavior or on the locomotor activity of the tested animals. Combinations of the antiepileptic drugs with either CHA (7 mg/kg, i.p.) or CGP 37849 (10 mg/kg, i.p.) also were devoid of adverse effects on the motor performance and long-term memory in mice demonstrated by the Chimney test and passive avoidance task. CHA (7 mg/kg, i.p.) and CGP 37849 (10 mg/kg, i.p.) didn’t affect the plasma level of any of the tested antiepileptic drugs. It could be concluded that adenosine A1 agonists and NMDA antagonists enhance the efficacy of common antiepileptic drugs. The potential therapeutic benefits of such interactions should be taken into consideration and merits further investigations in animals and humans.

Vancomycin exhibits multicompartment pharmacokinetics and demonstrates considerable interpatient variability in adults; however, only limited disposition data are available for children over one year of age.

PURPOSE: To characterize the vancomycin pharmacokinetic profile in individual pediatric patients and explore the potential of nonlinear mixed effects modeling and Bayesian forecasting for monitoring vancomycin in children.

METHODS: Serial blood samples obtained from six pediatric patients around the final steady-state vancomycin dose were analyzed by fluorescence polarization immunoassay. The vancomycin Ke, Vd, t1/2, and Cl were calculated for each patient based on a one-compartment open model by the 2-point (1) and Sawchuk and Zaske (2) methods. The vancomycin pharmacokinetic profile of each patient and population models were generated using NONMEM V (1.1). Both one- and two-compartment models were compared. To obtain Bayesian estimates of individual pharmacokinetic parameters and vancomycin concentration predictions, the two-compartment population parameters, interindividual and intraindividual variances, and each patient’s measured serum concentration were fixed in NONMEM. 

RESULTS: Mean pharmacokinetic estimates of t1/2, Vd and Cl derived by the 2-point (1) method were 3.52 h, 0.63 L/kg and 0.13 L/h/kg, respectively; whereas, those calculated by the Sawchuk and Zaske (2) method were 3.52 h, 0.57 L/kg and 0.12 L/h/kg, respectively. NONMEM analyses of the individual serial sampling data demonstrated that a weight-adjusted two-compartment model described the data better than a comparable one-compartment model, with mean objective function values of –12.67 and 138.67, respectively. The mean pharmacokinetic parameter estimates of t1/2 alpha, t1/2 beta, Vss, and Cl were 0.80 h, 5.63 h, 0.63 L/kg, and 0.11 L/h/kg, respectively. NONMEM population modeling also revealed that the weight-adjusted two-compartment model provided a better fit than a comparable one-compartment model, with objective function values of 99.32 and 192.46, respectively. The final two-compartment model generated population values of 0.77 h, 5.29 h, 0.65 L/kg, and 0.10 L/h/kg for t1/2 alpha, t1/2 beta, Vss, and Cl, respectively. Bayesian estimation using a two-compartment model and single midinterval serum samples indicated that accurate and precise predictions of vancomycin peak and trough concentrations could be obtained. Moreover, trough sampling alone, when incorporated into a Bayesian routine, provided clinically acceptable predictions of peak and post-distributional serum concentrations. 

CONCLUSIONS: Both individual and population pharmacokinetic modeling demonstrated that a two-compartment model provided a superior fit to the data. Based upon the means of individual estimates, the t1/2 alpha, t1/2 beta, Vss, and Cl were markedly different from those reported by Schaad et al (3). The observed differences can largely be explained by underestimation of the AUC by Schaad et al (3). The relatively long t1/2 alpha suggests that peak vancomycin concentrations measured earlier than four hours post-dose do not reflect post-distributional serum concentrations. It should be possible to simplify vancomycin monitoring by using trough samples or possibly, residual material from routinely obtained samples.

The effect of co-administration of magnesium sulfate and calcium channel blockers (nifedipine , verapamil) on the analgesic effect of opioid (morphine) and non-opioid drugs (acetylsalicylic acid: ASA). Albino mice and rats were used as experimental animals. Analgesia was measured in a hot-plate (52.5 degrees C), tail-flick (radiant heat source), formalin and acetic acid tests. The pain threshold was evaluated before and after the co-administration (intraperitoneal) of the different agents. Magensium sulfate (50 microgram/gm, i.p.), nifedipine and

verapamil (100 microgram/gm, i.p.) enhanced the analgesic effect of morphine sulfate (20-40 microgram/gm) in all tests. These agents ,however, failed to significantly increase the analgesic effect of ASA (50 microgram/gm, i.p.).

Conclusion: the co-administraion of calcium channel blockers and magnesium sulfate potentiate the analgesic effect of morphine but not that of ASA, and merts further studies in animals and humans.

Purpose:The Malay Peninsula though endowed with 9000 vascular plant species, utilizes only 1200 species in traditional medicine. Thus the flora of the Malay Peninsula still remain as vast untapped reservoirs for exploration and screening of plants for pharmacologically active molecules. The currently available anticancer drugs are associated with severe side effects and high mortality rates. Antibiotics such as the penicillins are becoming ineffective and many cases of resistance against methicillin are already reported. Most of the smooth muscle acting drugs used today are indeed costly synthetic molecules and are still far from being free of secondary side effects. Thus the plant kingdom with more than half a million species may provide the vital lead molecules for future anticancer, antimicrobial and smooth muscle acting drugs.

Method:With this purpose in mind 50 ethanolic/water extracts were prepared from 27 medicinal plant species collected from the virgin forests of Perak, Malaysia. These extracts were screened for the first time for their in vitro cytotoxic activity using a colorimetric assay to determine cell survival of human epidermoid (mouth) carcinoma KB cellls at 100 µg/ml. Similarly 10 µl of aquous extract (100 µg/ml) were tested for their smooth muscle activity on guinea pig ileum mounted in Tyrode’s solution. Relaxing or contracting effects were measured with the polygraph. For antibacterial screening the ethanolic extracts of leaves, barks, roots and whole plant were tested for in vitro activity against Staphylococcus aureus and Escherichia coli.

Introduction: The benefits and pitfalls associated with the relationship between a large Contract Research Organization (CRO) and a small innovator, biotechnology or generic pharmaceutical enterprise are explored from a Project Management standpoint using four areas of focus: regulatory and scientific expertise, risk management, joint ventures and the "one-stop shop" paradigm.

Purpose: To gain a better appreciation of the Project Management needs and expectations of the small R&D enterprise from Phoenix International Life Sciences, a multinational CRO. The information presented also intends to dispel some of the myths and assumptions that haunt and possibly jeopardize the initiation of this unique and potentially rewarding relationship.

Methods: Phoenix’s experience demonstrates overall trends in the unique and often surprising dependencies that small firms place on a CRO’s Project Management capabilities. Information gathered from small R&D firms with respect to their concerns in dealing with large CRO’s are presented.

Results: The results of our analysis points to a clear need for the Project Manager to address specific regulatory issues proactively. At any given time, large CRO’s specializing in global research services in all phases of drug discovery and development are exposed to a vast array of international regulatory agencies and their policies. Over time, this accumulated experience can be both a boon and an abrupt reality dose to small firms that may be encountering the regulatory mine field for the first time. The information collected from small innovator and biotech firms points to a greater dependency on our scientific expertise than from the generic counterparts. Consequently, Phoenix ‘s approach to staffing the Project Management team has resulted in drawing individuals with diverse scientific backgrounds from within its own operational groups, the industry and academia. The perceived risks impending over the relationship between the small firm and Phoenix are explored from either side. The results demonstrate the need for multiple-contingency planning at several levels from financial stability to scheduled deliverables. The growing trend of joint ventures between small firms and big pharma presents our contract research firm with the challenges and opportunities in Project Management of combining a familiar approach with a more creative plan to accommodate 2 partners and successfully be a part of that collaboration. The different approaches to out-sourcing often used by small R&D firms, namely the one-stop shop and diversified out-sourcing strategies present very different challenges to Project Management. Our experience shows that as a large CRO, Phoenix adds value when we are flexible enough to manage both strategies. This is facilitated by dedicating one Project Manager across all work for that sponsor to ensure consistency.

Conclusion: The Project Management challenges and opportunities inherent in the relationships between small R&D companies and a large CRO have been brought to the forefront. This has resulted in a greater appreciation of the unique needs of these relationships and the discovery that certain foregone assumptions about the relationships can be questioned.

Purpose : The "classic" nonprescription antihistamine diphenhydramine has been shown to interact with the polymorphic cytochrome P450 enzyme CYP2D6. This study was designed to investigate whether this interaction also manifests as a clinically significant alteration of the pharmacodynamics of the beta1-selective antagonist metoprolol in vivo.

Methods : 16 subjects with genetically high (extensive metabolizers) and 4 with low (poor metabolizers) CYP2D6 activity received a single dose of 100 mg metoprolol on the 3rd day of a 5 day course of either placebo or diphenhydramine administered thrice daily. The placebo/diphenhydramine administration was done in a double blind, cross over and randomized fashion. Resting and exercising blood pressure, heart rate and Doppler derived hemodynamic parameters were assessed for a period of 12 hours following metoprolol administration. Complete urine collection was done and plasma samples were collected at predetermined times for a period of 48 hours.

Results : Diphenhydramine decreased metoprolol oral and nonrenal clearances and metoprolol->alpha hydroxymetoprolol partial metabolic clearance in extensive metabolizers (EMs) but not in poor metabolizers (PMs). The metoprolol-related effects on heart rate and systolic blood pressure were more pronounced and lasted longer in EMs receiving diphenhydramine compared with EMs receiving placebo. Additionally, these effects were more pronounced in the PMs compared to the EMs. Diphenhydramine administration in the EMs was also shown to alter the VTI compared to the EMs administered placebo. The hemodynamic effects to metoprolol were unaltered in PMs administered diphenhydramine compared to the poor PMs administered placebo. Results from other doppler derived hemodynamic parameters measured such as stroke volume, cardiac index, peak acceleration and peak velocity are currently being compiled and will be presented during the conference.

Conclusions : Diphenhydramine has been shown to inhibit the metabolism of metoprolol in EMs, thereby prolonging the negative chronotropic and ionotropic effects of the drug. Clinically relevant drug interactions may occur between diphenhydramine and CYP2D6 substrates, particularly those presenting a narrow therapeutic index.

Purpose: Pravastatin, a HMG-CoA reductase inhibitor, is a therapeutic drug used for the reduction of cholesterol. The objective of this study is to develop and validate a high-throughput micro-extraction LC/MS/MS method for reliable routine measurement of pravastatin in human plasma in support of clinical investigations.

Methods:A micro-extraction procedure for pravastatin was developed using a 96-well solid-phase micro-extraction system. Plasma (1 ml) containing pravastatin was loaded into previously conditioned 96 well micro-extraction wells, washed with an aqueous buffer and eluted into autosampler vials for direct LC/MS/MS analysis. Mevastatin was used as an internal standard. LC/MS/MS analysis was carried out in +ESI mode using a triple quadrupole mass spectrometer system. Quantitation of pravastatin was carried out using multiple-reaction-monitoring (MRM) at m/z 424.5>327.2. Liquid chromatographic conditions were optimized using a gradient solvent system containing ammonium acetate and acetonitrile. Validation of the method over the plasma concentration range from 0.1 to 10 ng/ml involved examination of precision, accuracy, range, linearity, specificity, stability, recovery, quantitation limit and robustness of the method in reference to the ICH guidelines (1995) for method validation. The validation studies were scheduled over 5 separate analytical batches performed by two analysts over 3 working days.

Results: The micro-extraction procedure provided a reliable procedure for extraction of plasma samples into autosampler vial for direct LC/MS/MS analysis. The procedure required minimal manual handling of samples and thus provided added reliability and reproducibility. Plasma quality control samples and 0.1, 1 and 10 ng/ml and calibration standards at 0.1, 0.2, 0.5, 1, 2, 5 and 10 ng/ml were observed to provide a linear and reliable quantitation over the calibration range. The pravastatin MRM data was observed to be free from chromatographic and mass spectral interference. The stability of pravastatin in plasma during freeze/thaw and storage below –20C was demonstrated for up to 28 days. The extracted plasma sample was also observed to be stable for 24 hours at the autosampler. The 96-well micro-extraction procedure was observed to provide a mean extraction recovery of 76%. The lower limit of quantitation of the method was validated at 0.1 ng/ml assaying 1 ml of plasma.

Conclusion: A high-throughput 96-well micro-extraction LC/MS/MS method has been successfully developed and validated for reliable routine measurement of pravastatin in human plasma. This method has provided advantages in a lower quantitation limit and the procedure is free from manual sample handling compared over existing GC/MS methods which require an elaborate chemical derivatization procedure. The current micro-extraction LC/MS/MS method will allow the preparation and LC/MS/MS analysis of 200 plasma samples including standard calibration and QC samples with relative ease by one analyst. The method can further be adapted for fully an automated liquid handling autosampler or robotic system to allow continuous uninterrupted operation.

Purpose and Background: The combination of excipients with active materials, or compounding, in pharmaceutical formulations is a practise which has been synonymous with the development of modern pharmacy. These excipient materials have been referred to as ‘inactive ingredients’ and their traditional function has been to act as an inert vehicle for the active compound, diluents, lubricants, disintegrants etc.. It has long been realised, however, that certain materials that do not, in themselves, possess a pharmaceutical activity are able to significantly alter the effectiveness of ‘active compounds’ when they are present in combination. Towards the end of the 80’s a paradigm shift occurred in the industry with the introduction of high-throughput screening methods based upon combinational chemistry which matched compounds to molecular targets. The overall effect of this has been the production of compounds with a high specific activity, but with poor water solubility. This has meant that many promising compounds demonstrate a poor bioavailability when delivered by the oral route, the most popular form of drug administration. This has created the need for drug delivery technologies that enhance bioavailability. It has for some time been apparent, that lipid materials are able to affect the way in which drugs are absorbed by the body. Thus, excipient manufacturers specialising in this area have the potential to turn their products into drug delivery technologies and themselves become drug delivery companies.

Methods: It has been possible to develop formulation systems which represent optimal forms of lipid-based delivery system. Through following a defined formulations programme, using specific excipient blends it is possible to build systems around candidate active compounds which convert to optically clear microemulsions on contact with the gastrointestinal tract. The drug is present in molecular solution in a nanodispersed system. This has led to the creation of a proprietary technology SMEDDS – Self Microemulsifying Drug Delivery Systems. This methodology is described.

Results: The key parameters in defining successful formulation of lipid-based systems are physical and chemical stability, an acceptable drug loading, the production of an acceptable and stable dosage form. In the production of self-microemulsifying systems the particle size of the dispersed system is also a key parameter, since it is in this form that water-insoluble drug substances are presented to the gastro-intestinal tract in an optimal form for absorption.

Conclusion: Formulations of this type are very attractive in terms of scale-up and production, since the process involves little more than a simple mixing of the components and filling into capsules. This simplicity is beguiling, however, and more properly represents the conclusion of a more complex formulations programme. In order to ensure that a formulation based upon these principles is to be successful many parameters need to be considered and controlled: the loading level of the drug in the system; the particle size of the resulting emulsion; the biopharmaceutical class of the compound; the physical and chemical stability of the system, both before and after conversion to an emulsified system. These problems and their solutions are discussed in the light of the results.

Purpose: Heat-induced superaggregation of Amphotericin B (AmB) has been previously shown to reduce its in vitro and in vivo toxicity. The purpose of this study was to examine the interaction of this superaggregated form of AmB with serum lipoproteins.

Results:

Lipoprotein distribution studies show that for HFZ compared to FZ controls, a significantly lower percentage of AmB was recovered in the HDL, LDL, and TRL fractions, while a greater percentage of AmB was recovered in the LPD fraction. CD spectroscopy studies indicate that the "core" aggregate of HFZ is more stable in the presence of HDL and LDL, whereas the core aggregate of FZ dissociates to a greater extent in the presence of HDL and LDL.

Conclusions: These findings suggest that heat-induced superaggregation of AmB modifies its interaction with HDL and LDL, which may be important in explaining the increased therapeutic index of the superaggregated form of AmB.

Acknowledgements. This project was funded by the Medical Research Council of Canada (grant # MT-14484) and the National Science Foundation (MCB-9603582 to SCH). EHK was supported with a studentship from the Heart and Stroke Foundation of B.C. & Yukon.

Purpose. To develop a highly sensitive gas chromatographic/mass spectrometric (GS/MS) technique in order to profile and quantitate low levels of valproic acid (VPA) hydroxylated metabolites generated from the micro-incubation systems of cDNA-expressed or hepatic microsomal human cytochrome P450 enzymes. Methods. A microextraction procedure was developed to isolate VPA metabolites from small incubation volumes (100 ul) of human recombinant or hepatic microsomal cytochrome P450 enzymes. Extracted metabolites were treated with pentafluorobenzyl bromide to derivatize carboxyl groups, an essential step for the negative ion chemical ionization (NICI) GC/MS. A second step derivatization with N-methyl-N-tert-butyldimethylsilyl) trifluoroacetamide/tert-butyldimethylsilyl chloride (70 degree C for three hr) was introduced to silylate free hydroxyl groups of 3-hydroxy, 4-hydroxy, and 5-hydroxy-VPA. This sharpened the peaks and enhanced the signal to noise ratio of hydroxylated metabolites. A full separation of fifteen VPA metabolites, including all mono and diunsaturated VPA metabolites, was achieved by using a narrow-bore non-polar DB-1 column (0.25 mm X 60 m X 0.25 um, J. & W. Scientific). To maintain the run-time short (<28 min), a new temperature gradient was introduced with slow gradient at 120 to 160 degree C range followed by fast gradient to 270 degree C. Results. Double derivatization of hydroxylated VPA metabolites and analysis in NICI mode enhanced sensitivity significantly (L.O.D.: 0.2-20 pmol/ml). The derivatives of mono- and di-unsaturated metabolites, like the parent drug, produced abundant [M-181]-ions. The hydroxylated metabolites gave an ion m/z 273, corresponding to the [M-181]-ion of the tert-butyldimethylsilyl derivatives. The method was validated and a good precision and accuracy (intra- and inter-assay %relative error and %coefficient of variation < 10%) were obtained for all valproate metabolites in linear standard curve range. The method was applied successfully to conduct the P450 reaction phenotyping of valproic acid hydroxylated and unsaturated metabolites. Conclusions. The use of the inherent soft ionization nature of electron-capture NICI and double derivatization of hydroxylated VPA metabolites enabled us to achieve the high sensitivity necessary to conduct kinetic studies using small amounts of recombinant human P450 enzymes.

Acknowledgments. Supported by Medical Research Council of Canada.