Early-life caffeine exposure induces morphological changes and altered physiology in Caenorhabditiselegans.

Caffeine, a widely consumed stimulant, is known for its effects on alertness and fatigue reduction by blockade of adenosine receptors. While it holds therapeutic potential, its diverse impacts pose risks, particularly in early development. This study explores the developmental effects of caffeine exposure using Caenorhabditis elegans (C. elegans) as a model organism. We investigated morphological and behavioral changes induced by caffeine exposure at the L1 stage and assessed their impact at the L4 stage, which roughly corresponds to human infancy and adolescence, respectively. Caffeine-exposed worms displayed increased body length, body bends, and pharyngeal pumping rates compared to control worms. These findings indicate heightened food-seeking behavior and greater food intake, leading to the observed morphological changes. While caffeine did not affect other locomotor behaviors, its stimulatory effect on growth and development highlights its significance. This study provides insights into the potential impact of early-life caffeine exposure on long-term health and development, offering a foundation for future research in vertebrates to uncover its implications on metabolism and other metrics of health.

Student perceptions of scientific writing in pharmacology: Student generation of collaborative rubrics to score a social pharmacology writing project.

Scholarly writing is an important skill in all fields of study. Despite a strong focus on writing in many courses, faculty and students have disparate expectations related to scholarly writing. Herein, a classroom exercise is presented in which students were asked to develop a rubric that would be used to evaluate their summative writing assessment. Students were provided with a list of elements that commonly represent good scholarly writing, asked to define what effectively demonstrating these elements looks like, and asked to assign the weight that would be given to each element. The weights given to each element by students were compared to a faculty-generated, departmental writing rubric. Students assigned significantly higher weights to ideas, and significantly lower weights to sentence fluency. Overall, students favored content over writing mechanics. A random selection of student papers was scored using both the departmental rubric and the student rubric, with about a half-letter grade difference between the two groups, though the difference was not statistically significant. The outcomes suggest this exercise may be valuable in offering insight into student perceptions of scholarly writing and in furthering student engagement in the writing process.

Effects of a recombinant humanized anti-cocaine monoclonal antibody on the metabolism and distribution of cocaine in vitro and in mice.

The anti-cocaine monoclonal antibody, h2E2, is a candidate for treating cocaine-use disorder. h2E2 binds to and sequesters cocaine in the plasma compartment, effectively decreasing cocaine concentrations in the brains of rats and mice. Despite the binding of cocaine to h2E2, plasma cocaine concentrations decline rapidly in rodents over time, but there was a drastic decrease in the urinary elimination of cocaine in the presence of h2E2. Since cocaine is not being renally excreted, the apparent disappearance of cocaine from the plasma must be explained by either metabolism or distribution. However, binding of cocaine to h2E2 may restrict the availability of cocaine for hydrolysis by endogenous esterases. Therefore, the antibody would be expected to extend the elimination half-life of cocaine. In contrast, previous studies reported h2E2 as having no effect on the rate of cocaine clearance. It is important to examine the ultimate clearance of the cocaine to ascertain its half-life and potential for re-intoxication. Therefore, we investigated the effects of h2E2 on cocaine hydrolysis in vitro and on cocaine metabolism and disposition in vivo over a 6-h time course. The spontaneous and enzyme-mediated in vitro hydrolysis of cocaine was drastically decreased in the presence of h2E2 in vitro. Additionally, in mice, h2E2 significantly increased the distribution and elimination half-lives of cocaine relative to vehicle controls over an extended time course. Therefore, we concluded that h2E2 slowing the distribution and elimination of cocaine is the most appropriate explanation for the initial disappearance of cocaine from the plasma in vivo.

Differential effects of acute and chronic antagonist and an irreversible antagonist treatment on cocaine self-administration behavior in rats.

According to pharmacological theory, the magnitude of an agonist-induced response is related to the number of receptors occupied. If there is a receptor reserve, when the number of receptors is altered the fractional occupancy required to maintain this set number of receptors will change. Therefore, any change in dopamine receptor number will result in a change in the concentration of cocaine required to induce the satiety response. Rats that self-administered cocaine were treated with the irreversible monoamine receptor antagonist, EEDQ, or were infused continuously for 14 days with the D1-like antagonist, SCH23390, treatments known to decrease or increase, respectively, the number of dopamine receptors with a concomitant decrease or increase in response to dopaminergic agonists. The rate of cocaine maintained self-administration increased or decreased in rats treated with EEDQ or withdrawn from chronic SCH23390 infusion, respectively. After EEDQ treatment, the effect ratio of a single dose of SCH23390 or eticlopride were unchanged, indicating that the same SCH23390- and eticlopride-sensitive receptor populations (presumably dopamine) mediated the accelerated cocaine self-administration. Changing the receptor reserve is a key determinant of the rate of cocaine self-administration because the resulting increased or decreased concentration of cocaine results in an accelerated or decelerated rate of cocaine elimination as dictated by first-order kinetics.

The compulsion zone explains the self-administration of cocaine, RTI-55 and bupropion in rats.

Rats that reliably self-administered cocaine also reliably self-administered the cocaine analog RTI-55 and bupropion. The inter-injection intervals of these dopamine transporter (DAT) inhibitors were regular at a given unit dose and increased as a function of unit dose. However, the mean rate of intake differed widely, ranging from 731 to 459 to 2.1 nmol/kg∙min-1 for bupropion, cocaine and RTI-55 respectively, a dramatic 348-fold range. An analysis of inter-injection intervals as a function of unit dose generated values for the mean satiety threshold of 50.6, 5.1 and 0.7 nmol/kg and t1/2 of 56.7, 9.3 and 255.6 min for bupropion, cocaine and RTI-55, respectively. The difference in rate of intake of bupropion and RTI-55 relative to cocaine is a product of their 0.1 and 7.3 fold difference in PD potency and their 6.1 and 27.5 fold difference in t1/2. Additionally, the relative durations of lever-pressing following termination of drug access correlated with the t1/2 estimates. It is hypothesized this duration represents the time required for the drug concentration to fall from the satiety threshold below the priming threshold (the minimum DAT inhibitor level that will induce lever-pressing). This indicates that the time needed for an animal to cease lever pressing following termination of access to the DAT inhibitor is predominately a function of the PK properties of the agonist. The self-administration behavior paradigm in the context of the compulsion zone theory can be used as a bioassay to determine the PK/PD properties of indirect dopamine receptor agonists.

A novel differential scanning fluorimetry analysis of a humanized anti-cocaine mAb and its ligand binding characteristics.

An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinities. Here, we used differential scanning fluorimetry (DSF) to characterize the stability and ligand binding properties of this antibody and its cocaine-binding Fab fragment. The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic. However, by using the "rotor" dye, DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide), which measures the rotational restriction of the fluorescent dye (as opposed to the Sypro orange dye which measures the hydrophobicity of the dye microenvironment), a simple two state thermal denaturation transition that is stabilized by ligand binding was observed for the h2E2 mAb, enabling Boltzmann fitting and quantitative thermodynamic analysis of the DASPMI DSF mAb cocaine and metabolite binding data. The computed affinities were consistent with ligand binding affinities determined using other techniques. Thus, this novel DASPMI DSF method can simply, inexpensively, and very rapidly generate ligand binding constants for the h2E2 mAb, despite the presence of multiple, overlapping, thermally unfolding protein domains characteristic of all mAbs. This approach is likely applicable to other mAbs currently in use for many research and therapeutic applications.

A Recombinant Humanized Anticocaine Monoclonal Antibody Alters the Urinary Clearance of Cocaine and Its Metabolites in Rats.

A recombinant humanized anticocaine monoclonal antibody, h2E2, has shown potential in the preclinical phases for the treatment of cocaine abuse. The standard tests for cocaine usage are the detection of benzoylecgonine (BE) and cocaine in the urine. This includes workplace drug screens as well as in clinical trials for potential treatments of cocaine abuse. By sequestering cocaine into the plasma compartment, h2E2 prevents cocaine from entering the brain. Due to the altered disposition of cocaine in the presence of h2E2, we investigated the effects of h2E2 on cocaine and metabolite levels in the urine of rats to clarify the use of BE as an endpoint measurement for effectiveness in future clinical trials. The urine concentrations of cocaine and metabolites were considerably altered in the presence of h2E2. After a single injection of h2E2 (120 mg/kg) and cocaine hydrochloride (0.56 mg/kg), the concentration of cocaine and BE excreted into the urine of rats decreased by 92% and 91%, respectively, from vehicle controls. Due to the significant decrease in urinary excretion, BE is not an appropriate indicator of cocaine usage in the presence of h2E2. Another endpoint measurement must be selected for the measurement of cocaine usage in the upcoming clinical trials of h2E2. In contrast to the effects on cocaine and BE urinary excretion, there was a 3-fold increase in ecgonine methyl ester (EME) in the presence of h2E2. Therefore, we conclude that EME is a more appropriate measurement of cocaine intake in the presence of h2E2.

Evaluation of methods to reduce background using the Python-based ELISA_QC program.

Almost all immunological approaches [immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blot], that are used to quantitate specific proteins have had to address high backgrounds due to non-specific reactivity. We report here for the first time a quantitative comparison of methods for reduction of the background of commercial biotinylated antibodies using the Python-based ELISA_QC program. This is demonstrated using a recombinant humanized anti-cocaine monoclonal antibody. Several approaches, such as adjustment of the incubation time and the concentration of blocking agent, as well as the dilution of secondary antibodies, have been explored to address this issue. In this report, systematic comparisons of two different methods, contrasted with other more traditional methods to address this problem are provided. Addition of heparin (HP) at 1 µg/ml to the wash buffer prior to addition of the secondary biotinylated antibody reduced the elevated background absorbance values (from a mean of 0.313 ±â€¯0.015 to 0.137 ±â€¯0.002). A novel immunodepletion (ID) method also reduced the background (from a mean of 0.331 ±â€¯0.010 to 0.146 ±â€¯0.013). Overall, the ID method generated more similar results at each concentration of the ELISA standard curve to that using the standard lot 1 than the HP method, as analyzed by the Python-based ELISA_QC program. We conclude that the ID method, while more laborious, provides the best solution to resolve the high background seen with specific lots of biotinylated secondary antibody.

A mathematical model of a recombinant humanized anti-cocaine monoclonal antibody's effects on cocaine pharmacokinetics in mice.

AIMS: A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood. MAIN METHODS: A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over 1h in the presence and absence of h2E2. KEY FINDINGS: The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment. SIGNIFICANCE: This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs.

A novel Python program for implementation of quality control in the ELISA.

The use of semi-quantitative assays such as the enzyme-linked immunosorbent assay (ELISA) requires stringent quality control of the data. However, such quality control is often lacking in academic settings due to unavailability of software and knowledge. Therefore, our aim was to develop methods to easily implement Levey-Jennings quality control methods. For this purpose, we created a program written in Python (a programming language with an open-source license) and tested it using a training set of ELISA standard curves quantifying the Fab fragment of an anti-cocaine monoclonal antibody in mouse blood. A colorimetric ELISA was developed using a goat anti-human anti-Fab capture method. Mouse blood samples spiked with the Fab fragment were tested against a standard curve of known concentrations of Fab fragment in buffer over a period of 133days stored at 4°C to assess stability of the Fab fragment and to generate a test dataset to assess the program. All standard curves were analyzed using our program to batch process the data and to generate Levey-Jennings control charts and statistics regarding the datasets. The program was able to identify values outside of two standard deviations, and this identification of outliers was consistent with the results of a two-way ANOVA. This program is freely available, which will help laboratories implement quality control methods, thus improving reproducibility within and between labs. We report here successful testing of the program with our training set and development of a method for quantification of the Fab fragment in mouse blood.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.

The effects of a repeated dose of a recombinant humanized anti-cocaine monoclonal antibody on cocaine self-administration in rats.

BACKGROUND: Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested. METHODS: The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3µmol/kg and 3µmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120mg/kg i.v.) 35days apart. Priming threshold and inter-injection intervals were measured for 35days after both injections. RESULTS: After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35days. A significant decrease (15-33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 infusions at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection. CONCLUSIONS: These data predict that the safety and effectiveness of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse.

Cytotoxicity and apoptotic gene expression in an in vitro model of the blood-brain barrier following exposure to poly(butylcyanoacrylate) nanoparticles.

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear. Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood-brain barrier. Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array. Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control. Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity. Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.

The effects of a humanized recombinant anti-cocaine monoclonal antibody on the disposition of cocaethylene in mice.

The chimeric human/mouse anti-cocaine monoclonal antibody (mAb) 2E2 and its further humanized variant h2E2 have been reported to sequester a significant portion of cocaine in plasma and decrease cocaine concentrations in the brain in mice and rats. However, many cocaine users co-abuse alcohol, leading to the formation of the centrally active metabolite cocaethylene. This potentially compromises the efficacy of a cocaine-specific immunotherapy. Because h2E2 has high affinity for cocaethylene as well as cocaine, the ability of h2E2 to prevent cocaethylene entry into the brain was investigated. Mice were infused with h2E2 (1.6 µmol/kg i.v.) or vehicle and after one hour were injected with cocaethylene fumarate (1.2 µmol/kg i.v.). At times from 45 s to 60 min, brain and plasma were collected and cocaethylene concentrations were measured using GC/MS. In control mice, a two-compartment pharmacokinetic model generated values for cocaethylene distribution and terminal elimination half-lives of 0.5 and 8.1 min respectively. Initial plasma cocaethylene concentrations increased 13-fold from controls in the presence of h2E2. In brain, h2E2 produced a 92% decrease in the area under the time-concentration curve for cocaethylene. The pharmacokinetics of h2E2 was also characterized in detail. A three-compartment model resolved an initial distribution half-life of 4.4 min and a second distribution half-life of 4.2 h, and a terminal elimination half-life of 7.8 days. The ability of h2E2 to protect the brain from both cocaine and cocaethylene predicts that the clinical efficacy of h2E2 will be retained in cocaine users who co-abuse alcohol.