Microdosing and Other Phase 0 Clinical Trials: Facilitating Translation in Drug Development.

A number of drivers and developments suggest that microdosing and other phase 0 applications will experience increased utilization in the near-to-medium future. Increasing costs of drug development and ethical concerns about the risks of exposing humans and animals to novel chemical entities are important drivers in favor of these approaches, and can be expected only to increase in their relevance. An increasing body of research supports the validity of extrapolation from the limited drug exposure of phase 0 approaches to the full, therapeutic exposure, with modeling and simulations capable of extrapolating even non-linear scenarios. An increasing number of applications and design options demonstrate the versatility and flexibility these approaches offer to drug developers including the study of PK, bioavailability, DDI, and mechanistic PD effects. PET microdosing allows study of target localization, PK and receptor binding and occupancy, while Intra-Target Microdosing (ITM) allows study of local therapeutic-level acute PD coupled with systemic microdose-level exposure. Applications in vulnerable populations and extreme environments are attractive due to the unique risks of pharmacotherapy and increasing unmet healthcare needs. All phase 0 approaches depend on the validity of extrapolation from the limited-exposure scenario to the full exposure of therapeutic intent, but in the final analysis the potential for controlled human data to reduce uncertainty about drug properties is bound to be a valuable addition to the drug development process.

A microdose study of ¹4C-AR-709 in healthy men: pharmacokinetics, absolute bioavailability and concentrations in key compartments of the lung.

PURPOSE: To explore, in a microdose (phase-0) study, the pharmacokinetics, bioavailability and concentrations in key compartments of the lung, of AR-709, a novel diaminopyrimidine antibiotic for the treatment of respiratory infection. METHODS: Four healthy men each received two single, 100 µg microdoses of ¹4C-AR-709, 7 days apart: the first was administered intravenously (IV), the second orally. Plasma pharmacokinetics of ¹4C and unchanged AR-709 were obtained by high-performance liquid chromatography and accelerator mass spectrometry (AMS). Next, 15 healthy men received a single, 100 µg microdose of ¹4C-AR-709 IV. Plasma, bronchoalveolar lavage fluid, alveolar macrophages and bronchial mucosal biopsy samples were analysed by AMS. RESULTS: After IV administration, clearance of AR-709 was 496 mL/min, volume of distribution was 1,700 L and the absolute oral bioavailability was 2.5 %. Excretion in urine was negligible. At 8-12 h after IV dosing, ¹4C concentrations in lung samples were 15- (bronchial mucosa) to 200- (alveolar macrophages) fold higher than in plasma. In alveolar macrophages, ¹4C was still mostly associated with AR-709 at 12 h after dosing. CONCLUSIONS: The results of this microdose study indicate that AR-709 attains concentrations appreciably higher within the lung than in plasma. Its low oral bioavailability however, precludes oral administration. Although IV administration would appear to be an effective route of administration, this would limit the use of AR-709 to a clinical setting and would therefore be economically unsustainable. If further clinical development were to be undertaken, therefore, an alternative route of administration would be necessary.

Development of 2D chiral chromatography with accelerator mass spectrometry for quantification of (14)C-labeled R- and S-verapamil in plasma.

BACKGROUND: A microdose study was performed where 50 µg R/S-(14)C-verapamil was dosed intravenously to human volunteers. In order to quantify the individual R- and S-enantiomers in human plasma a 2D chiral HPLC method with subsequent analysis by accelerator mass spectrometry was verified. RESULTS: R/S-verapamil was separated on a C18 column and the isolated fraction was applied to a chiral column where the verapamil enantiomers were separated. Experimental recovery (∼73% [coefficient of variation {CV} = 16%] and 66% [CV = 21%] for R- and S-verapamil, respectively) was accounted for by the use of internal standardization from the fluorescence response of nonlabeled R- and S-verapamil. The precision of the assay ranged from 4.1 to 15.9% CV and the limit of quantitation was 1.95-4.81 pg/ml for R-verapamil and 1.76-3.34 pg/ml for S-verapamil. CONCLUSION: This method was successfully applied to the analysis of R- and S-verapamil in human plasma.

Investigator-initiated trials of targeted oncology agents: why independent research is at risk?

BACKGROUND: Drug development traditionally has relied upon the complementary contributions of clinicians and scientists at academic institutions and at pharmaceutical companies. Greater regulatory burdens, increased bureaucratic requirements, restricted reimbursement, and spiralling research and development costs are exerting pressure on the drug development pipeline. The result is a de-emphasis of exploratory research, particularly independent academic research, despite its proven value in identifying new drug targets and developing innovative cancer therapies. DESIGN: An expert panel assembled by the Biotherapy Development Association-a nonprofit international forum for academic and industry researchers, patients, and government regulatory and postregulatory agencies-examined the growing schism between academia and industry and identified several causes of declining academic research. RESULTS: The authors propose solutions to sustain investigator-initiated research and provide a new model whereby expert organisations provide a forum for academia and industry to plan studies within a regulatory framework to support licensure/authorisation and reimbursement for new molecularly targeted agents and biomarkers. CONCLUSIONS: Investigator-initiated trials have led to the discovery and development of innovative, safe, and effective cancer treatments. To ensure that such research continues, action will be required on the parts of legislative and regulatory bodies, industry, universities, patient advocacy organisations, and preclinical and clinical academic scientists.

A sensitive method for the determination of chlorine-36 in foods using accelerator mass spectrometry.

A method using accelerator mass spectrometry (AMS) has been developed to offer a more sensitive alternative to scintillation techniques for the determination of chlorine-36 ((36)Cl) in foods. The main problem in method development was the potential interference of the sulfur-36 ((36)S) isobar. This was overcome by reducing the sulfur level of acid digests of food by precipitation of chloride as silver chloride, then purification by washing, dissolution and reprecipitation to present silver chloride as the AMS target. The limit of detection was around 0.1 Bq kg(-1) and the limit of quantitation was around 0.2 Bq kg(-1). The AMS method was only semi-quantitative at the lowest levels of interest. To test the method a few samples of milk (five) and blackberries (three) collected from near two nuclear power stations as potential sources of contamination were analysed. Blackberries spiked at 0.2 Bq kg(-1) and milk spiked at 0.1 Bq kg(-1) could be distinguished from method blanks. There was no (36)Cl detectable in the unspiked samples.

High-performance liquid chromatography accelerator mass spectrometry: correcting for losses during analysis by internal standardization.

A method was developed to account for analytical losses of (14)C-analyte when determining the concentration in biological samples using chromatographic separation and analysis by accelerator mass spectrometry. From the equations of J. Vogel and A.H. Love (in: A.L. Burlingame (Ed.), Methods in Enzymology, Academic Press, New York, 2005), new equations were derived to describe the isotopic dilution of a chromatographically isolated (14)C-analyte. The analytical recovery for each sample was determined by the use of the UV response for nonlabeled analyte, as an internal standard against a standard curve. The slope of the curve was substituted into the equations to provide a method of accurately determining the analyte concentration.

Novel use of accelerator mass spectrometry for the quantification of low levels of systemic therapeutic recombinant protein.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

Determination of the bioavailability of [14C]-hexaminolevulinate using accelerator mass spectrometry after intravesical administration to human volunteers.

Hexaminolevulinate (HAL) is a diagnostic agent that allows the visualization of tumor tissue in the bladder by fluorescence cystoscopy. It is administered intravesically via a catheter for 1 hour, followed by blue light bladder inspection to induce selective red tumor fluorescence. Hexaminolevulinate should ideally be confined to the bladder only, but it is likely that some absorption occurs during administration, and therefore the systemic bioavailability is of interest. The bioavailability of HAL was determined by intravesical and intravenous administration of [14C]-HAL hydrochloride to 8 human volunteers. To reduce the radiation dose as low as possible, the ultrasensitive analytical technique of accelerator mass spectrometry was used to measure [14C]-HAL. The bioavailability of [14C]-HAL after intravesical and intravenous administration was determined from the respective area under the curve based on total radioactivity and was determined to be 7% (range, 5%-10%; 90% confidence interval). The systemic absorption of [14C]-HAL after intravesical administration is low and supports previous clinical experience with HAL showing no systemic side effects.

Comparative metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) in rat and dog.

1. There is a significant species difference in the toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D). The oral no overall adverse effect level (NOAEL) for chronic toxicity of 2,4-D in rat is 5 mg kg(-1) day(-1) and in dog is 1 mg kg(-1) day(-1). The maximum tolerated dose (MTD) in rat is 150 and 75 kg(-1) day(-1) for male and females, respectively. The MTD in dog is 7.5 mg kg(-1) day(-1) for males and females. 2. In an attempt to explain the increased sensitivity to 2,4-D in dog, male and female rats and dogs were orally dosed with either 5 or 50 mg kg(-1) 14C-2,4-D. The rates and routes of excretion were investigated along with plasma toxicokinetics and biotransformation of the compound. 3. Elimination of the radioactive dose of 2,4-D from rat plasma was significantly faster than in dog. The approximate t(1/2) were 1.3-3.4 h for rat and 99-134 h for dog following a 5 or 50 mg kg(-1) dose, respectively. This led to large differences in the calculated AUC(0-infinity) 21-57 microg eq. h g(-1) for rat and 4889-5298 microg eq. h g(-1) for dog at 5 mg kg(-1), and 122-2358 microg eq. h g(-1) for rat and 34,110-44,296 microg eq. h g(-1) for dog at 50 mg kg(-1)). 4. In rat, the major route of excretion was in the urine. Excretion was essentially complete after 24 h for the low dose and after 48 h for the high dose. For dog, elimination was incomplete over the sampling period with only about 50% of the dose recovered. Urine was the principal route of excretion at the low dose, but about equal amounts were excreted in urine and faeces at the high dose over 120 h. 5. In rat, 2,4-D was unmetabolized and excreted in urine as the parent compound. In dog, the dose was excreted mainly following metabolism. 2,4-D in dog was conjugated forming the taurine, serine, glycine, glutamic acid, cysteine, sulphate and glucuronide conjugates, plus an unidentified metabolite, which were excreted in urine. Plasma, however, only contained unmetabolized 2,4-D. 6. The results show that the body burden of 2,4-D in dog is significantly higher than in rat for an equivalent dose, which is consistent with the increased sensitivity of dog to 2,4-D toxicity.

Metabolism of methyl-(E)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl]-3-methoxyacrylate (azoxystrobin) in rat.

1. The metabolic fate of [(14)C]-methyl-(E)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl]-3-methoxyacrylate (azoxystrobin) was determined in the male and female rat following a single oral dose of 1 and 100 mg x kg(-1) and in surgically prepared, bile duct-cannulated rats following a single oral dose of 100 mg x kg(-1). 2. Azoxystrobin was extensively metabolized with at least 15 metabolites. There was a sex difference, with females producing more metabolites than males. 3. The two principal metabolic pathways were hydrolysis of the methoxyacid followed by glucuronic acid conjugation and glutathione conjugation of the cyanophenyl ring followed by further metabolism leading to the mercapturic acid. There were also several other minor pathways.

Absorption, metabolism and excretion of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat and dog.

1. The oral no overall adverse effect level (NOAEL) for chronic toxicity of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat is approximately 1.3 mg kg(-1) and in dog is 0.2 mg kg(-1). In an attempt to explain the difference in toxicology between these species, rats and dogs were orally dosed with (14C)-MCPA at 5 or 100 mgkg(-1) and plasma toxicokinetics, rates and routes of excretion and biotransformation were investigated. 2. Elimination of radioactivity in rat plasma was biphasic and in dog was monophasic. Rat eliminated radioactivity from plasma significantly faster than dog (approximate values biased on total radioactivity: 5 mg kg(-1) rat: t 1/2 dist 3.5 h, t 1/2 elim 17.2-36.2 h, AUC(0-infinity) 230 microg equiv hg(-1); 5 mg kg(-1) dog: t 1/2 47h, AUC(0-infinity) 2,500 microg equiv h g(-1); 100 mg kg(-1) rat: t 1/2 dist 10h, t 1/2 elim 10.27-25.4h, AUC(0-infinity) 5,400 microg equiv hg(-1); l00 mg kg(-1) dog: t 1/2 h, AUC(0-infinity) 20,500 microg eqiv h g(-1). 3. For both species, the principal route of excretion was in urine but renal elimination was notably more rapid and more extensive in rat. 4. In both rat and dog, excretion of radioactivity was mainly as MCPA and its hydroxylated metabolite hydroxymethylphenoxyacetic acid (HMCPA). In rat, both were mainly excreted as the free acids although a small proportion was conjugated. In dog, the proportion of HMCPA was increased and the majority of both species was excreted as glycine or taurine conjugates. 5. These data, along with previously published accounts, indicate that renal elimination of MCPA in dog is substantially slower than in rat resulting in disproportionate elevation of AUC (based on total radioactivity) in dog compared with rat.

Metabolism of 2,3,5,6-tetrachloronitrobenzene (tecnazene) in rat.

1. The metabolic fate of [U-14C]-2,3,5,6-tetrachloronitrobenzene (tecnazene) has been determined in the male and female rat following a single dose of 1 mg/kg and in surgically prepared, bile-duct-cannulated rats following a single oral dose of 135 mg/kg. 2. Radioactivity in the female rat was excreted mainly in urine (82%). The male rat, however, excreted approximately equal amounts of radioactivity in urine and faeces (the latter via bile). 3. The principal metabolic pathway was conjugation with glutathione (GSH) and concomitant nitro-displacement. The GSH-conjugate and related metabolites were excreted in the bile and ultimately in the urine as the mercapturic acid conjugate. The cysteine conjugate underwent beta-lyase-mediated metabolism to yield a thiol that underwent subsequent methylation to the thioanisole followed by S-oxidation. 4. A novel tetrachloromethyldisulphide metabolite was also formed.

The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study.

Repeated administration of high doses of captan is known to produce tumours specifically in the duodenum of mice. Captan is not carcinogenic in the rat. In this study, DNA purified from the liver, stomach, duodenum and jejenum of mice dosed with 35S radiolabelled captan was found to contain radioactivity equivalent to Covalent Binding Indices in the range 38-91; that from the bone marrow had a CBI of 2.8. The distribution of radioactivity between the various tissues did not reflect the target organ specificity of captan. Attempts to further purify the DNA samples using caesium chloride gradients resulted in partial separation of the radioactivity from the DNA suggesting that covalent binding to the DNA may not have occurred. A study of the chemical breakdown of captan showed that captan is unstable, producing a variety of potentially reactive species containing sulphur. Evidence was further obtained to show that the sulphur of captan is incorporated into endogenous amino acids and protein. Hepatic DNA from mice dosed with 35S radiolabelled N-acetylcysteine, and two thiazolidine derivatives which are analogous to known metabolites of captan, was radiolabelled to a similar extent to that from captan treated mice. Furthermore, the DNA from each of these treatments had similar properties on caesium chloride gradients. It was concluded that the radioactivity associated with DNA in the captan DNA binding study was present in the low levels of protein which are always associated with purified DNA samples.