News
researchers have only been able to use it twice. "We have not found the ultimate polymer yet," says Powell. "We need to increase its affinity for cAMP to make it more practical. It's a novel finding in that it is the first time that a ligand has actually been imprinted into a polymer that contains both recognition and fluorescence sensing elements." The approach may be a first step towards developing a biomimetic sensor for aqueous cAMP; however, there are still some areas that need improvement. "Once we come up with something that is a bit more sensitive to cAMP, then we can try the same techniques to look at other second messengers." Britt Erickson
Facile estimation of partitioning coefficients of drugs Drug design is big business, and in the quest for new bioactive compounds many synthetic advances have been made with combinatorial approaches. The large number of compounds consequently generated creates great demand for reliable characterization of their physicochemical properties. One of these properties is hydrophobicity (a measure of a compound's ability to partition through biological membranes), which is estimated by measurement of the thermodynamic octanol-water partition coefficient (log P). The octanol phase mimics the hydrophobic biomembrane. Until now, the standard method for accurate measurement of log P has been the time-consuming "shake-flask" technique. This method typically takes one day per compound a timescale unacceptable to drug discovery teams. Recent advances by Patrick Camilleri and co-workers at SmithKline Beecham Pharmaceuticals (U.K.) with collaborators Melissa Hanna and Andrew Hutt at King's College London could change all that. Camilleri considers that "log P is frequently the most significant physicochemical parameter routinely considered in drug design." He and his research team have focused on developing a means for its rapid and reliable estimation. Three years ago, these workers were the first to use micellar electrokinetic capillary chromatography (MECC) for the determination of partition coefficients for drugs, and in the May 15th issue of Analytical Chemistry (p. 2092) they extend that work by reporting a rapid method for the prediction of log P with
Buckyball
breath test A humidity sensor fast enough to follow human breathing has been developed by California chemists based on the ubiquitous buckyball. The tough new material is far more sensitive in the lowhumidity region than current solid-state sensors and could eventually be applied in several analytical environments. Galen Stucky and graduate student Andrew Saab at the University of California-Santa Barbara have found that they can mediate the conductivity of a derivative of the buckyball (properly known as [60]fullerene) by exposing it to water vapor. They have fabricated the compound into a durable thin film, which operates over the whole humidity range but is especially sensitive to the low levels of humidity required for opto-electronics fabrication, according to Stucky. Chemists have added all sorts of doping agents and groups to fullerenes in an effort to affect electrical properties, but practical applications are still limited. "Research by [our] group on the diffusion and transport properties of Cgo suggested that there might be some mileage in making an ionicaUy conducting material," says Stucky. "Subsequent efforts were directed toward controllably reproducing the water-sensitive films."
reversed-phase HPLC and MECC for a wide range of drugs (acidic, neutral, and basic). The new protocol "combines die best features of reversed-phase HPLC and MECC," says Camilleri. "The system can therefore provide a very reliable estimate of log P." The ability of this system to provide rapid screening of mixtures of potentially diverse compounds is of enormous benefit to those involved in drug discovery. Both the HPLC and MECC methods are based on die same fundamental principle as the shake-flask system, indeed they can be viewed as automated versions of the same. Each involves primarily the measurement of the extent to which the test compounds distribute between an aqueous and an hydrophobic phase. The more hydrophobic a compound, the greater the association with the hydrophobic phase, and hence the more it will be "retained". Retention parameters (such as the capacity factor, k') determined
372 A Analytical Chemistry News & Features, June 1, 1998
^he team beSan kv using water vapor and oxygen to slowly oxidize a previously described potassium-doped fullerene under an intense xenon light. The exact conditions were arrived at by trial and error, but once they had established a preparation method they set about testing the film's sensitivity to water vapor (Adv. Mater. 1998,10,462-65). To avoid polarization of the material, the researchers recorded AC conductivity in an atmosphere of pure water vapor covering the pressure range 0.03-25 mbar. This corresponds to a range of about 0.1-100% relative humidity. In the lowest region, 0-5 % relative humidity, ,he material is up to six times more sensitive than some conventional materials. Stucky says that although the UV-vis spectrum of the film is not very different from that of pure [60]fullerene, the near-IR photoacoustic response of the bulk material hints at the presence of highly functionalized species with hydrogen and oxygen atoms, and hydroxy groups. "The unique fullerene nature of the material could be mediating the conductivity," explains Stucky. He adds, "The material certainly has potential for humidity sensing applications, although it will require a further R&D effort aimed specifically at technical applications before the real usability of the material is known." David Bradley
by HPLC and MECC should therefore be correlated with log P values. A conceptual advance made in this work towards obtaining the best estimate of the in vivo behavior of potentially bioactive compounds is to design the hydrophobic phase to resemble as closely as possible a biomembrane. A possible means of achieving this goal is by use of a natural biomembrane component, such as phosphatidylcholine. Camilleri and co-workers prepare the stationary phase for HPLC by cycling a 1-mM solution of lecithin (phosphatidylcholine) in methanol-water through the column for 24 h. This method, Camilleri says, "may lead to die formation of multilayers" that allow biologically relevant partitioning of the test compounds. Columns made in this simple way are very stable and last for weeks without degradation. The MECC system incorporated phosphatidylcholine bile acid mixed micelles in the aqueous buffer.
Retention parameters determined by each method correlated well with shakeflask log P values. Combining the data from both techniques provides a better predictive model than does either method alone. Camilleri and co-workers demonstrated the method by analyzing 106 structurally diverse compounds (acids, bases, and neutrals) with a wide range of log P values (-0.52 to 7.63). Indeed, the system is so efficient that, rather than serving solely to characterize potential new drugs, the authors anticipate that it may also contribute to the design and selection of candidate drug substances.
Shake-flask technology still has its place in drug registration, where accurate values are required, but even here "it may eventually be superseded," says Camilleri. What's next? Efforts to modify the hydrophobic phase to even more closely mimic biomembranes are ongoing. Camilleri believes the next major advance will involve the incorporation of liposomes, containing varying amounts of different naturally occurring membrane lipids. Other potential uses can be envisaged in the field of agrochemicals and the screening of environmental pollutants. Raewyn Town
GOVERNMENT AND SOCIETY
NACLA launches membership drive
An open workshop was held on April 16 at NIST to discuss the status of the National Cooperation for Laboratory Accreditation (NACLA), a program aimed at reducing the number of accreditations for testing and calibration laboratories in the United States (Anal. Chem. 1197,69,161 A). Organizers of NACLA believe that if all accreditors are recognized by the same procedure, then accreditors and their accredited laboratories will all meet the same minimum level of performance. Since its NEWS FROM THE ACS NATIONAL MEETING establishment in May 1997, NACLA's interim board of directors has been developBritt Erickson reports from Dallas. ing procedures and criteria for accreditation and reciprocal recognition. Their ongophase-Ill clinical trial, which showed reImproved control ing efforts have resulted in the approval of sponse rates equivalent to chemotherapy methods lead to NACLA documents the bylaws, recognibut without the severe side effects, FDA faster drug approval approved Rituxan for the treatment of non- tion document, and quality manuall A membership drive is now underway to solicit "Therapeutic monoclonal antibodies initially Hodgkin's lymphoma in less than six got off to a slow start," says Robert Garnick, months. A lot can be learned by looking at interest in NACIA, with expectations that it will be operational by September 1998 the analytical methods that were used to vice president of regulatory affairs for Gecharacterize Rituxan, says Garnick. nentech, "due to production, control, and Laboratory accreditation in the United immunogenicity problems." However, tilings States has evolved into a complicated mixRituxan, which is a "humanized" chihave changed, and, according to Garnick, it meric monoclonal antibody, is an excellent ture of more than 150 different programs. is thought that approximately 80% of the new example of a molecule that could be conWith so many programs, it becomes a timebiologies waiting to be approved by the FDA sidered well-characterized. Multiple strucconsuming, costly, and at times confusing are monoclonal antibodies. task for laboratories to become recognized tural methods were used to confirm the identity of the compound, including Edman as competent, say NACLA organizers. Part of the problem is that high doses Moreover, there is no guarantee that a labsequencing and peptide mapping by MS. (0.5-4 g) of monoclonal antibodies are oforatory will also be recognized internationThe secondary structure was investigated ten required to achieve the desired theraally. According to Belinda Collins, director using circular dichroism. The purity of the peutic effect for cancer treatment. Those of NIST's Office of Standards Services and compound was determined based on high-dose requirements put a tremendous chair of NACLA's interim board accreditastrain on drug manufacturers who typically charge heterogeneity (ion-exchange CE tion typically costs a laboratory operating have to produce 50-200 kg of drug per year with isoelectric focusing), size-exclusion in the United States from $10 000 to chromatography, and sodium dodecyl sulto meet these needs. More importantly, $50,000 per year. fate polyacrylamide gel electrophoresis. because such large amounts of the drugs Potency testing was performed using a are required, they must be of the highest NACLA organizers say that they do highly biomimetic bioassay that mimics the not intend to become another accreditapurity. Historically, analytical methods for function of the drug in humans. the characterization of therapeutic monotion body but rather a coordinating body. clonal antibodies have focused on purity As a cooperative partnership between the Unexpectedly, the molecule was found and impurity determinations, but that is all to contain three forms of biantennary oligo- private and public sectors, NACLA could changing, says Garnick. Today quality con- saccharides, which all differed in their biolead to fewer accreditors and more worldtrol efforts are focusing on biological activ- logical activity. Glycosylation adds tremen- wide acceptance of ity and its relationship to carbohydrate discredible data. As dous complexity to a molecule, says Gartribution As a result of improved characthe program develnick. The three forms could be separated terization and control methods the FDA ops, it is expected by CE, and the bioactivity of the molecule approval of monoclonal antibodies has was found to be a function of galactose con- to broaden its membeen greatly facilitated and accelerated bership to include tent. Garnick believes that CE is an invaluaccreditation bodies For example, the rapid FDA approval of able tool for routine quality control of bioin Mexico and Cantechnology products. The recent advances the drug Rituximab, sold under the tradeada the NA in in CE and MS have significantly improved mark Rituxan, is a prime example of how Belinda Collins the control and characterization of theraattitudes at FDA are changing, says GarNACLA will eventu- solicits interest in peutic monoclonal antibodies, he says. nick. Based on a 166-patient open-labeled ally stand for North NACLA Analytical Chemistry News & Features, June 1, 1998 3 7 3 A
