Characterization of PF-6142, a Novel, Non-Catecholamine Dopamine Receptor D1 Agonist, in Murine and Nonhuman Primate Models of Dopaminergic Activation.

Selective activation of dopamine D1 receptors remains a promising pro-cognitive therapeutic strategy awaiting robust clinical investigation. PF-6142 is a key example from a recently disclosed novel series of non-catechol agonists and partial agonists of the dopamine D1/5 receptors (D1R) that exhibit pharmacokinetic (PK) properties suitable for oral delivery. Given their reported potential for functionally biased signaling compared to known catechol-based selective agonists, and the promising rodent PK profile of PF-6142, we utilized relevant in vivo assays in male rodents and male and female non-human primates (NHP) to evaluate the pharmacology of this new series. Studies in rodents showed that PF-6142 increased locomotor activity and prefrontal cortex acetylcholine release, increased time spent in wakefulness, and desynchronized the EEG, like known D1R agonists. D1R selectivity of PF-6142 was supported by lack of effect in D1R knock-out mice and blocked response in the presence of the D1R antagonist SCH-23390. Further, PF-6142 improved performance in rodent models of NMDA receptor antagonist-induced cognitive dysfunction, such as MK-801-disrupted paired-pulse facilitation, and ketamine-disrupted working memory performance in the radial arm maze. Similarly, PF-6142 reversed ketamine-induced deficits in NHP performing the spatial delayed recognition task. Of importance, PF-6142 did not alter the efficacy of risperidone in assays predictive of antipsychotic-like effect in rodents including pre-pulse inhibition and conditioned avoidance responding. These data support the continued development of non-catechol based D1R agonists for the treatment of cognitive impairment associated with brain disorders including schizophrenia.

Antibody Fragment F(ab')2 Targeting Caveolae-Associated Protein PV1 for Selective Kidney Targeting and Retention.

Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.

Bioengineering of bacterial pathogens for noninvasive imaging and in vivo evaluation of therapeutics.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2'deoxy-2'-fluoro-ß-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [18F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [18F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥107 bacteria per infection site. This was further verified by administering [18F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.

Evaluation of Volume of Intramuscular Injection into the Caudal Thigh Muscles of Female and Male BALB/c Mice (Mus musculus).

This study presents recommendations for intramuscular injection into the caudal thigh muscle of mice according to analysis of in vivo imaging of intramuscularly injected iohexol, a radiocontrast agent commonly used in CT imaging. An experienced laboratory animal technician using a Hamilton syringe intramuscularly injected iohexol into isoflurane-anesthetized female and male BALB/c mice. Injected volumes (25, 50, 100, and 200 µL) underwent CT scanning at 9 time points over a 3-h period. The distribution of the injectate in the muscles of the rear leg was examined over time for each volume group. Results indicated that 25- and 50-µL volumes remain intramuscularly. At 100 µL, mild to moderate leakage into the extramuscular tissues occurred. At 200 µL, leakage into the extramuscular tissues was moderate to severe. Our results suggest volumes of 50 µL or less are recommended for the caudal thigh muscles of mice when intramuscular pharmacokinetics are needed; volumes greater than 50 µL display variable distribution into extramuscular tissues, thus potentially yielding different pharmacokinetic profiles.

[18F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice.

PURPOSE: The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [18F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice. PROCEDURES: We assessed ZIKV-induced pathogenesis in wild-type C57BL/6 mice administered an antibody to inhibit type I interferon (IFN) signaling. [18F]DPA-714 PET imaging was performed on days 3, 6, and 10 post-infection (PI), and tissues were subsequently processed for histological evaluation, quantification of microgliosis, and detection of viral RNA by in situ hybridization (ISH). RESULTS: In susceptible ZIKV-infected mice, viral titers in the brain increased from days 3 to 10 PI. Over this span, these mice showed a two- to sixfold increase in global brain neuroinflammation using [18F]DPA-714 PET imaging despite limited, regional detection of viral RNA. No measurable increase in ionized calcium binding adaptor molecule 1 (Iba-1) expression was noted at day 3 PI; however, there was a modest increase at day 6 PI and an approximately significant fourfold increase in Iba-1 expression at day 10 PI in the susceptible ZIKV-infected group relative to controls. CONCLUSIONS: The results of the current study demonstrate that global neuroinflammation plays a significant role in the progression of ZIKV infection and that [18F]DPA-714 PET imaging is a sensitive tool relative to histology for the detection of neuroinflammation. [18F]DPA-714 PET imaging may be useful in dynamically characterizing the pathology associated with neurotropic viruses and the evaluation of therapeutics being developed for treatment of infectious diseases.

The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring Radioligand for Positron Emission Tomography (PET) Imaging.

As part of our effort in identifying phosphodiesterase (PDE) 4B-preferring inhibitors for the treatment of central nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to enable target occupancy measurement in vivo. Through a systematic and cost-effective PET discovery process, involving expression level (Bmax) and biodistribution determination, a PET-specific structure-activity relationship (SAR) effort, and specific binding assessment using a LC-MS/MS "cold tracer" method, we have identified 8 (PF-06445974) as a promising PET lead. Compound 8 has exquisite potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of specific binding in the "cold tracer" study. In subsequent non-human primate (NHP) PET imaging studies, [18F]8 showed rapid brain uptake and high target specificity, indicating that [18F]8 is a promising PDE4B-preferring radioligand for clinical PET imaging.

Neuropathogenesis of Zika Virus in a Highly Susceptible Immunocompetent Mouse Model after Antibody Blockade of Type I Interferon.

Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection.

The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH).

INTRODUCTION: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [(18)F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain. METHODS: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [(18)F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [(18)F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1h prior to radiotracer injection. RESULTS: Biodistribution studies show good uptake (SUV~0.8 at 90 min) of [(18)F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%-73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [(18)F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845. CONCLUSIONS: [(18)F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.

[(18)F]FLT-PET imaging does not always "light up" proliferating tumor cells.

PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.

PF-03732010: a fully human monoclonal antibody against P-cadherin with antitumor and antimetastatic activity.

PURPOSE: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. EXPERIMENTAL DESIGN: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. RESULTS: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane ß-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. CONCLUSIONS: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials.

Evaluation of treatment-associated inflammatory response on diffusion-weighted magnetic resonance imaging and 2-[18F]-fluoro-2-deoxy-D-glucose-positron emission tomography imaging biomarkers.

PURPOSE: Functional imaging biomarkers of cancer treatment response offer the potential for early determination of outcome through the assessment of biochemical, physiologic, and microenvironmental readouts. Cell death may result in an immunologic response, thus complicating the interpretation of biomarker readouts. This study evaluated the temporal effect of treatment-associated inflammatory activity on diffusion magnetic resonance imaging and 2-[(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography imaging (FDG-PET) biomarkers to delineate the effects of the inflammatory response on imaging readouts. EXPERIMENTAL DESIGN: Rats with intracerebral 9L gliosarcomas were separated into four groups consisting of control, an immunosuppressive agent dexamethasone (Dex), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and BCNU+Dex. Animals were imaged using diffusion-weighted magnetic resonance imaging and FDG-PET at 0, 3, and 7 days posttreatment. RESULTS: In the BCNU- and BCNU+Dex-treated animal groups, diffusion values increased progressively over the 7-day study period to approximately 23% over baseline. The FDG percentage change of standard uptake value decreased at day 3 (-30.9%) but increased over baseline levels at day 7 (+20.1%). FDG-PET of BCNU+Dex-treated animals were found to have percentage of standard uptake value reductions of -31.4% and -24.7% at days 3 and 7, respectively, following treatment. Activated macrophages were observed on day 7 in the BCNU treatment group with much fewer found in the BCNU+Dex group. CONCLUSIONS: Results revealed that treatment-associated inflammatory response following tumor therapy resulted in the accentuation of tumor diffusion response along with a corresponding increase in tumor FDG uptake due to the presence of glucose-consuming activated macrophages. The dynamics and magnitude of potential inflammatory response should be considered when interpreting imaging biomarker results.

PF-00477736 mediates checkpoint kinase 1 signaling pathway and potentiates docetaxel-induced efficacy in xenografts.

PURPOSE: Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736-mediated modulation of biomarkers and the sensitization of docetaxel efficacy. EXPERIMENTAL DESIGN: In vitro and in vivo studies using COLO205 and other cell lines were done to assess PF-00477736-induced enhancement of docetaxel efficacy and effects on associated biomarkers. RESULTS: PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser(345)), phosphorylated histone H3 (Ser(10)), and gammaH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser(216)). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser(216)) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [18F]FLT-PET imaging. However, changes in [18F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival. CONCLUSIONS: Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.

Proton and sodium MRI assessment of emerging tumor chemotherapeutic resistance.

The ultimate goal of any cancer therapy is to target the elimination of neoplastic cells. Although newer therapeutic strategies are in constant development, therapeutic assessment has been hampered by the inability to assess, rapidly and quantitatively, efficacy in vivo. Diffusion imaging and, more recently, sodium MRI have demonstrated their distinct abilities to detect therapy-induced alterations in tumor cellularity, which has been demonstrated to be indicative of therapeutic efficacy. More importantly, both imaging modalities detect tumor response much earlier than traditional methodologies that rely on macroscopic volumetric changes. In this study, the correlation between tumor sodium and diffusion was further tested to demonstrate the sensitivity of sodium imaging to gauge tumor response to therapy by using a 9L rat gliosarcoma treated with varying doses of BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea]. This orthotopic model has been demonstrated to display variability in response to BCNU therapy where initial insult has been shown to lead to drug-resistance. In brief, a single 26.6 mg/kg BCNU dose yielded dramatic responses in both diffusion and sodium MRI. However, a second equivalent BCNU dose yielded a much smaller change in diffusion and sodium, suggesting a drop in tumor sensitivity to BCNU. The MRI responses of animals treated with 13.3 mg/kg BCNU were much lower and similar responses were observed after the initial and secondary applications of BCNU. Furthermore, these results were further validated using volumetric measurements of the tumor and also ex vivo determination of tumor sensitivity to BCNU. Overall, these experiments demonstrate the sensitivity and applicability of sodium and diffusion MRI as tools for dynamic assessment of tumor response to therapy.

A methodology for registration of a histological slide and in vivo MRI volume based on optimizing mutual information.

We present a method for registering histology and in vivo imaging that requires minimal microtoming and is automatic following the user's initialization. In this demonstration, we register a single hematoxylin-and-eosin-stained histological slide of a coronal section of a rat brain harboring a 9L gliosarcoma with an in vivo 7T MR image volume of the same brain. Because the spatial resolution of the in vivo MRI is limited, we add the step of obtaining a high spatial resolution, ex vivo MRI in situ for intermediate registration. The approach taken was to maximize mutual information in order to optimize the registration between all pairings of image data whether the sources are MRI, tissue block photograph, or stained sample photograph. The warping interpolant used was thin plate splines with the appropriate basis function for either 2-D or 3-D applications. All registrations were implemented by user initialization of the approximate pose between the two data sets, followed by automatic optimization based on maximizing mutual information. Only the higher quality anatomical images were used in the registration process; however, the spatial transformation was directly applied to a quantitative diffusion image. Quantitative diffusion maps from the registered location appeared highly correlated with the H&E slide. Overall, this approach provides a robust method for coregistration of in vivo images with histological sections and will have broad applications in the field of functional and molecular imaging.

Sodium and proton diffusion MRI as biomarkers for early therapeutic response in subcutaneous tumors.

The ability to quantitate early effects of tumor therapeutic response using noninvasive imaging would have a major impact in clinical oncology. One area of active research interest is the ability to use MR techniques to detect subtle changes in tumor cellular density. In this study, sodium and proton diffusion MRI were compared for their ability to detect early cellular changes in tumors treated with a cytotoxic chemotherapy. Subcutaneous 9L gliosarcomas were treated with a single dose of 1,3-bis(2-chloroethyl)-1-nitrosourea. Both sodium and diffusion imaging modalities were able to detect changes in tumor cellularity as early as 2 days after treatment, which continued to evolve as increased signal intensities reached a maximum approximately 8 days posttreatment. Early changes in tumor sodium and apparent diffusion coefficient values were predictive of subsequent tumor shrinkage, which occurred approximately 10 days later. Overall, therapeutical induced changes in sodium and diffusion values were found to have similar dynamic and spatial changes. These findings suggest that these imaging modalities detected similar early cellular changes after treatment. The results of this study support the continued clinical testing of diffusion MRI for evaluation of early tumor treatment response and demonstrate the complementary insights of sodium MRI for oncology applications.

In vivo measures of dopaminergic radioligands in the rat brain: equilibrium infusion studies.

The application of an equilibrium infusion method for measuring specific in vivo radioligand binding in the conscious rat brain was evaluated for two ligands of the dopaminergic system, (+)-alpha-[(11)C]dihydrotetrabenazine (DTBZ) and d-threo-[(11)C]methylphenidate (MePhen). Both radioligands can be successfully utilized to reach equilibrium distributions in rat brain within 1 h; combinations of tritiated and carbon-11-labeled radiotracers can furthermore be used to obtain simultaneous measures of the neuronal membrane dopamine transporter (using [(3)H]MePhen) and vesicular monoamine transporter (using [(11)C]DTBZ) in the same animal. These studies provided quantitative measures of distribution volume ratios, which represent specific radioligand binding. Stereospecificity of in vivo binding was demonstrated using equilibrium infusions of the low-affinity isomers of each ligand, (-)-alpha-[(11)C]dihydrotetrabenazine (DTBZ) and l-threo-[(11)C]methylphenidate, both of which produced uniform brain distributions and no specific binding. Specific binding of (+)-alpha-[(11)C]dihydrotetrabenazine was blocked by co-infusion of tetrabenazine, but was unaffected by administration of methylphenidate, haloperidol, or apomorphine. Specific binding of d-threo-[(11)C]methylphenidate, conversely, was blocked with unlabeled methylphenidate but not affected by tetrabenazine or the dopamine receptor ligands. Equilibrium measures of in vivo radioligand binding, as utilized in this study, offer a quantitative means to evaluate acute and chronic drug effects on in vivo radioligand binding in the rat brain.