Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

Exploring the use of pharmaceutical patient assistance programs as an option to improve access to and affordability of prescription drugs

Background. Pharmaceutical-sponsored patient assistance programs (PAPs) are charity programs that provide free or reduced-priced medications to eligible patients. PAPs have the potential to improve prescription drug accessibility for patients but currently there is limited information about their use and effectiveness. ^ Objectives and methods. This dissertation described the use of PAPs in the U.S. through the conduct of two studies: (1) a systematic review of primary studies of PAPs from commercially-published and “grey” literature sources; and (2) a retrospective, cross-sectional study of cancer patients' use of PAPs at a tertiary care cancer outpatient center. ^ Results. (1) The systematic review identified 33 studies: 15 evaluated the impact of PAP enrollment assistance programs on patient healthcare outcomes; 7 assessed institutional costs of providing enrollment assistance; 7 surveyed stakeholders; 4 examined other aspects. Standardized mean differences calculated for disease indicator outcomes (most of which were single group, pre-posttest designs) showed significant decreases in glycemic and lipid control, and inconsistent results for blood pressure. Grey literature abstracts reported insufficient statistics for calculations. Study heterogeneity made weighted summary estimates inappropriate. Economic analyses indicated positive financial benefits to institutions providing enrollment assistance (cost) compared to the wholesale value of the medications provided (benefit); analyses did not value health outcomes. Mean quality of reporting scores were higher for observational studies in commercially-published articles versus full text, grey literature reports. (2) The cross-sectional study found that PAP outpatients were significantly more likely to be uninsured, indigent, and < 65 years old than non-PAP patients. Nearly all non-PAP and PAP prescriptions were for non-cancer conditions, either for co-morbidities (e.g., hypertension) or the management of treatment side effects (e.g., pain). Oral chemotherapies from PAPs were significantly more likely to be for breast versus other cancers, and be a newer, targeted versus traditional chemotherapy.^ Conclusions. In outpatient settings, PAP enrollment assistance plus additional medication services (e.g., counseling, reminders, and free samples) is associated with improved disease indicators for patients. Healthcare institutions, including cancer centers, can offset financial losses from uncompensated drug costs and recoup costs invested in enrollment assistance programs by procuring free PAP medications. Cancer patients who are indigent and uninsured may be able to access more outpatient medications for their supportive care needs through PAPs, than for cancer treatment options like oral chemotherapies. Because of the selective availability of drugs through PAPs, there may be more options for newer, oral, targeted chemotherapies for the treatment breast cancer versus other for other cancers.^

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Cell Cycle Regulatory Roles of Estrogen Receptor alpha (ERα) in Breast Cancer Cells

Previous studies have shown that Estrogen Receptor alpha (ERα) is an important indicator for diagnosis, prognosis and treatment of breast cancers. However, the question remains as to the role of ERα in the cell in the presence versus absence of 17-β estradiol In this dissertation the role of ERα in both its unliganded and liganded state, with respect to the cell cycle will be explored. The cell line models used in this project are ER-positive MCF-7 cells with and without siRNA to ERα and ER-positive MDA-MB-231 cells that have been engineered to express ERα. Cells were synchronized and the cell cycle progression was monitored by flow cytometric analysis. Using these methods, two specific questions were addressed: Does ERα modulate the cell cycle differently under liganded versus unliganded conditions? And, does the presence of ERα regulate cell cycle phase transitions? The results show for the first time that ERα is cell cycle regulated and modulates the progression of cells through S and G2/M phases of the cell cycle. Ligand bound ERα increases progression through S and G2/M phases, whereas unliganded ERα acts as an inhibitor of cell cycle progression. To further investigate the cell cycle regulated effects of liganded ERα, a luciferase assay was performed and showed that the transcription of target genes such as Progestrone Receptor (PgR) and Trefoil protein (pS2) increased duing S and G2/M phases when ERα is bound to ligand. Additionally, complex formation between cyclin B and ER α was shown by immunoprecipitation and led to the discovery that anaphase promoting complex (APC) is the E3 ligase for both cyclin B and ERα at the termination of M phase. Our findings suggest that unliganded ERα has an inhibitory effect on the progression of the cell cycle. Therefore, it is reasonable to speculate that the combination of drugs that lower estrogen level (such as aromatase inhibitors) and preserves ERα from degradation would provide better outcome for breast cancer treatment. We have shown that APC functions as the E3 ligase for ERα and thus might provide a target to design a specific inhibitor of ERα degradation.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Characteristics and experiences of past participants in the Texas Peer Assistance Program for Nurses

There are nearly 200,000 licensed practicing nurses in the state of Texas, representing one-tenth of the nations' workforce. The prevalence of substance abuse among nurses is estimated to range between six and 20 percent in this professional group.^ Since March 1987, the Texas Peer Assistance Program for Nurses (TPAPN) has offered intervention, education, support and monitoring to nurses in Texas whose practice has become impaired due to substance abuse and/or mental illness. Since then approximately 44 percent of nurses who voluntarily signed participation agreements successfully completed the program; fifty-six percent have not. One determinant of completion for those nurses identified as chemically dependent is abstinence from mood altering substances. Other helping professions report higher rates of abstinence two years following treatment.^ The purpose of this study was to investigate the relationship between relapse, demographics, treatment variables, work setting, "stress" indicators and support factors for nurses who participated in TPAPN. A questionnaire was mailed to 1000 randomly selected nurses who had signed agreements since 1987 and were no longer active in the program. More than 41% of the questionnaires were returned undeliverable.^ Recipients of the questionnaire were known only to TPAPN, never to the investigator. All information was received anonymously except when the participant chose to sign the questionnaire. A cover letter explaining the study and inviting participation was enclosed. Completion and return of the questionnaire was considered consent to participate.^ Findings demonstrated a significant relationship between relapse and opiates as the drug of choice for past participants in the Texas Peer Assistance Program for Nurses. Significant associations were found among factors such as control at work, support, physical complaints, job security, self-esteem and employment in this sample. Respondents shared copious written comments about their experiences in TPAPN. These data were analyzed using qualitative methods and compared with similar studies of recovering nurses. Further research with nurses whose practice has been affected by abuse of chemical and mental illness is warranted. ^

Childhood sexual victimization: Risk factor for drug use and sexual risk behaviors

The purpose of this dissertation was to survey men in the Harris County Jail (HCJ) to establish a more valid estimate of childhood sexual abuse (CSA) prevalence in a jailed-based population; to assess whether inmates with a history of CSA were at greater risk for use of drugs and alcohol and engaging in high-risk sexual behaviors than those without histories of childhood sexual abuse. ^ The first study determined the prevalence of childhood sexual abuse among incarcerated males in a county jail. In this study, sixty-three percent of the subjects reported having been sexually abused. Sixty-one percent reported abuse pre-puberty and 10% reported abuse post puberty. In pre-puberty abuse the initiation of first abuse occurred at a mean age of 5.6 years (SD 5.096, range: 2–13 years). ^ The second study explored the association between inmates with histories of CSA as a risk factor for sexual risk behaviors. A history of sexual abuse did not appear to be associated with an elevated risk of sexual risk behaviors. ^ The third study explored a history of drug use and a history of CSA among the inmates. A chi-square test showed that the inmates who reported a history of CSA, was significantly greater for the following drugs: Marijuana (02), Crack (03), Heroin/Morphine (.03), Amphetamines/Speed (01), Downers/Barbiturates (.001), Methamphetamine/Crystal Meth (.001), Valium .02), LSD/Acid (.001), and Inhalants (.001), p < .05). Significance was not found in alcohol, tobacco, cocaine, Quaaludes and methadone. ^ The research from this study provides empirical data supporting previous research. The current data shows that incarcerated inmates have a high prevalence of childhood sexual abuse and drug use. Sexual victimization as a child does not appear to be associated with an elevated risk of unsafe sexual behaviors. However, men who used drugs were twice as likely to have engaged in unprotected sex with casual and regular partners, and rarely used condoms with paid sex. Although our study methods do not permit a causal explanation for this association, we believe it is of concern. Finally, data in this study shows that sexually abused children are likely candidates for adult criminal behavior. ^

New cytochromes P450: Tale of two variants

Cytochrome P450s, a superfamily of heme enzymes found in most living organisms. They are responsible for metabolism of many therapeutic drugs, industrial pollutants, carcinogens, and additives to foodstuffs, as well as some endogenous compounds including fatty acids and steroids. First pass drug metabolism studies represent mainly liver and small intestine elimination, and are viewed as the standard to predict therapeutic outcome. However, drug plasma levels determined after administration do not always correlate with therapeutic efficacy of the drug. Therefore, a possible explanation may come by understanding drug metabolism in extrahepatic tissues and/or at the site of drug action. Identification and characterization of novel tissue specific isoforms of P450 generated by alternative splicing of known P450 genes or as yet unidentified genes is essential to predict pharmacological outcome of drugs or the fate of a carcinogen that act at sites remote from liver. ^ Using RT-PCR, brain-specific cytochrome P450s were detected in samples of human autopsy brain. So far, we have identified two human brain variants including P450 2D7 and P450 1A1. We have shown the presence of the P450 1A1 brain specific splice variant in African Americans, Caucasians and Indians albeit different patterns of liver to brain variant ratio were seen distributed throughout each population. Interestingly, the splice variant was detected only in the brain but not in any other tissues from the same individual. Homology modeling was used to compare the variant 3D structure to the liver form structure and differences in the substrate access channels and substrate binding sites were noticed. Automated computational docking was used to predict the metabolic fate of the potent carcinogenic substrate, benzo[a]pyrene. P450 1A1 brain variant showed no binding orientations that could produce the active metabolite, whereas P450 1A1 liver form did reveal orientations capable of generating active carcinogenic product. In vitro P32 labeling studies verified the docking predictions. Therefore, the data support the hypothesis that P450 brain splice variants mediate the metabolism of xenobiotics by mechanisms distinct from the well-studied liver counterparts. ^

Preclinical modeling of multi-drug cancer therapies

Anticancer drugs typically are administered in the clinic in the form of mixtures, sometimes called combinations. Only in rare cases, however, are mixtures approved as drugs. Rather, research on mixtures tends to occur after single drugs have been approved. The goal of this research project was to develop modeling approaches that would encourage rational preclinical mixture design. To this end, a series of models were developed. First, several QSAR classification models were constructed to predict the cytotoxicity, oral clearance, and acute systemic toxicity of drugs. The QSAR models were applied to a set of over 115,000 natural compounds in order to identify promising ones for testing in mixtures. Second, an improved method was developed to assess synergistic, antagonistic, and additive effects between drugs in a mixture. This method, dubbed the MixLow method, is similar to the Median-Effect method, the de facto standard for assessing drug interactions. The primary difference between the two is that the MixLow method uses a nonlinear mixed-effects model to estimate parameters of concentration-effect curves, rather than an ordinary least squares procedure. Parameter estimators produced by the MixLow method were more precise than those produced by the Median-Effect Method, and coverage of Loewe index confidence intervals was superior. Third, a model was developed to predict drug interactions based on scores obtained from virtual docking experiments. This represents a novel approach for modeling drug mixtures and was more useful for the data modeled here than competing approaches. The model was applied to cytotoxicity data for 45 mixtures, each composed of up to 10 selected drugs. One drug, doxorubicin, was a standard chemotherapy agent and the others were well-known natural compounds including curcumin, EGCG, quercetin, and rhein. Predictions of synergism/antagonism were made for all possible fixed-ratio mixtures, cytotoxicities of the 10 best-scoring mixtures were tested, and drug interactions were assessed. Predicted and observed responses were highly correlated (r2 = 0.83). Results suggested that some mixtures allowed up to an 11-fold reduction of doxorubicin concentrations without sacrificing efficacy. Taken together, the models developed in this project present a general approach to rational design of mixtures during preclinical drug development. ^

The impact of 1995--1996 health sector reforms in the effectiveness of malaria control program in the state of Orissa, India

Malaria poses a significant public health problem worldwide. The World Health Organization indicates that approximately 40% of the world's population and almost 85% of the population from the South–East Asian region is at risk of contracting malaria. India being the most populous country in the region, contributes the highest number of malaria cases and deaths attributed to malaria. Orissa is the state that has the highest number of malaria cases and deaths attributable to malaria. A secondary data analysis was carried out to evaluate the effectiveness of the World bank-assisted Malaria Action Program in the state of Orissa under the health sector reforms of 1995-96. The secondary analysis utilized the government of India's National Anti Malaria Management Information System's (NAMMIS) surveillance data and the National Family Health Survey (NFHS–I and NFHS–II) datasets to compare the malaria mortality and morbidity in the state between 1992-93 and 1998-99. Results revealed no effect of the intervention and indicated an increase of 2.18 times in malaria mortality between 1992-1999 and an increase of 1.53 times in malaria morbidity between 1992-93 and 1998-99 in the state. The difference in the age-adjusted malaria morbidity in the state between the time periods of 1992-93 and 1998-99 proved to be highly significant (t = 4.29 df=16, p<. 0005) whereas the difference between the increase of age-adjusted malaria morbidity during 1992-93 and 1998-99 between Orissa (with intervention) and Bihar (no intervention) proved to be non significant (t=.0471 df=16, p<.50). Factors such as underutilization of World Bank funds for the malaria control program, inadequate health care infrastructure, structural adjustment problems, poor management, poor financial management, parasite resistance to anti-malarial drugs, inadequate supply of drugs and staff shortages may have contributed to the failure of the program in the state.^

Frequencies and risk factors for bisphosphonate-related osteonecrosis of the jaw in cancer patients: A matched case-control study

Bisphosphonates represent a unique class of drugs that effectively treat and prevent a variety of bone-related disorders including metastatic bone disease and osteoporosis. High tolerance and high efficacy rates quickly ranked bisphosphonates as the standard of care for bone-related diseases. However, in the early 2000s, case reports began to surface that linked bisphosphonates with osteonecrosis of the jaw (ONJ). Since that time, studies conducted have corroborated the linkage. However, as with most disease states, many factors can contribute to the onset of disease. The aim of this study was to determine which comorbid factors presented an increased risk for developing ONJ in cancer patients.^ Using a case-control study design, investigators used a combination of ICD-9 codes and chart review to identify confirmed cases of ONJ at The University of Texas M. D. Anderson Cancer Center (MDACC). Each case was then matched to five controls based on age, gender, race/ethnicity, and primary cancer diagnosis. Data querying and chart review provided information on variables of interest. These variables included bisphosphonate exposure, glucocorticoids exposure, smoking history, obesity, and diabetes. Statistical analysis was conducted using PASW (Predictive Analytics Software) Statistics, Version 18 (SPSS Inc., Chicago, Illinois).^ One hundred twelve (112) cases were identified as confirmed cases of ONJ. Variables were run using univariate logistic regression to determine significance (p < .05); significant variables were included in the final conditional logistic regression model. Concurrent use of bisphosphonates and glucocorticoids (OR, 18.60; CI, 8.85 to 39.12; p < .001), current smokers (OR, 2.52; CI, 1.21 to 5.25; p = .014), and presence of diabetes (OR, 1.84; CI, 1.06 to 3.20; p = .030) were found to increase the risk for developing ONJ. Obesity was not associated significantly with ONJ development.^ In this study, cancer patients that received bisphosphonates as part of their therapeutic regimen were found to have an 18-fold increase in their risk of developing ONJ. Other factors included smoking and diabetes. More studies examining the concurrent use of glucocorticoids and bisphosphonates may be able to strengthen any correlations.^

The two -state model of receptor activation: The agonist and the efficacy

The Two State model describes how drugs activate receptors by inducing or supporting a conformational change in the receptor from “off” to “on”. The beta 2 adrenergic receptor system is the model system which was used to formalize the concept of two states, and the mechanism of hormone agonist stimulation of this receptor is similar to ligand activation of other seven transmembrane receptors. Hormone binding to beta 2 adrenergic receptors stimulates the intracellular production of cyclic adenosine monophosphate (cAMP), which is mediated through the stimulatory guanyl nucleotide binding protein (Gs) interacting with the membrane bound enzyme adenylylcyclase (AC). ^ The effects of cAMP include protein phosphorylation, metabolic regulation and transcriptional regulation. The beta 2 adrenergic receptor system is the most well known of its family of G protein coupled receptors. Ligands have been scrutinized extensively in search of more effective therapeutic agents at this receptor as well as for insight into the biochemical mechanism of receptor activation. Hormone binding to receptor is thought to induce a conformational change in the receptor that increases its affinity for inactive Gs, catalyzes the release of GDP and subsequent binding of GTP and activation of Gs. ^ However, some beta 2 ligands are more efficient at this transformation than others, and the underlying mechanism for this drug specificity is not fully understood. The central problem in pharmacology is the characterization of drugs in their effect on physiological systems, and consequently, the search for a rational scale of drug effectiveness has been the effort of many investigators, which continues to the present time as models are proposed, tested and modified. ^ The major results of this thesis show that for many b2 -adrenergic ligands, the Two State model is quite adequate to explain their activity, but dobutamine (+/−3,4-dihydroxy-N-[3-(4-hydroxyphenyl)-1-methylpropyl]- b -phenethylamine) fails to conform to the predictions of the Two State model. It is a weak partial agonist, but it forms a large amount of high affinity complexes, and these complexes are formed at low concentrations much better than at higher concentrations. Finally, dobutamine causes the beta 2 adrenergic receptor to form high affinity complexes at a much faster rate than can be accounted for by its low efficiency activating AC. Because the Two State model fails to predict the activity of dobutamine in three different ways, it has been disproven in its strictest form. ^