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To harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Here, model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drug would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native secondary structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that better recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to better maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy.

Immunogenic cell death (ICD) is a way of reengaging the tumor-specific immune system. ICD can be induced by treatment with chemotherapeutics. However, only a limited number of drugs and other treatment modalities have been shown to elicit the biomarker responses characteristic of ICD and to provide an anticancer benefit in vivo. Here, we report a rationally designed redox-active Au(I) bis-N-heterocyclic carbene that induces ICD both in vitro and in vivo. This work benefits from a synthetic pathway that allows for the facile preparation of asymmetric redox-active Au(I) bis-N-heterocyclic carbenes.

Acyclovir is a poorly permeable, short half-life drug with poor colonic absorption, and current conventional controlled release formulations are unable to decrease the frequency of administration. We designed acyclovir dosage forms to be administered less frequently by being retained in the stomach and releasing drug over an extended duration. We developed a conventional modified-release matrix tablet to sustain the release of acyclovir and surrounded it with a hydrophilic poly(urethane) layer. When hydrated, the porous poly(urethane) swells to a size near or beyond that of the relaxed pylorus diameter and does not affect drug release rate. We demonstrated that the formulation is retained in the stomach for extended durations as it slowly releases drug, allowing for similar area under the curve but delayed tmax relative to a nongastroretentive control tablet. Unlike many other gastroretentive formulations, this dosage form design decouples drug release rate from gastric retention time, allowing them to be modulated independently. It also effectively retains in the stomach regardless of the prandial state, differentiating from other approaches. Our direct observation of excised rat stomachs allowed for a rigorous assessment of the impact of polymer swelling extent and the prandial state on both the dosage form integrity and retention time.

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase ATM using a metabolism-centered CRISPR/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In the TCGA pan-cancer datasets, we found that ATM levels strongly correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-h pharmacokinetic study in hamsters demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 h. The half-life of GS-4412524 following dry powder insufflation was about 7 h, suggesting the dosing regimen would be twice-daily administration. Although the remdesivir-Captisol® (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease., Graphical abstract Unlabelled Image

In this work, we have developed and tested in vivo a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters. The niclosamide dry powder, suitable for inhalation, is being developed as a therapeutic agent against COVID-19 infection. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with a specific pharmacological effect against SARS-CoV-2 infection. In the past, clinical trials for other indications were terminated prior to completion due to low and highly variable oral bioavailability. In order to quickly address the current pandemic, targeting niclosamide directly to the lungs is rational to address the COVID-19 main clinical complications. Thin-film freezing technology was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 μm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose pharmacokinetic study in rats as evidenced by histopathology analysis, but also was able to achieve lung concentrations above the required IC50 and IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation formulation by thin-film freezing for further scale-up and clinical testing against the COVID-19 infection. This approach overcomes niclosamide’s limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-hour pharmacokinetic study in hamsters, a small animal model for SARS-CoV-2, demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 hours. The half-life of GS-4412524 following dry powder insufflation was about 7 hours, suggesting the dosing regimen would be twice daily administration. Although the remdesivir-Captisol® (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 μm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-β1 (Ad-TGF-β1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases.

Idiopathic pulmonary fibrosis (IPF) is associated with progressive destruction of the lung parenchyma with a median survival after diagnosis of three years and a five-year survival of

In this work, we have developed and tested a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters to gather data about its toxicology and pharmacokinetics. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with pharmacological effect against SARS-CoV-2 infection. TFF was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 µm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose tolerability and exposure study in rats as evidenced by histopathology analysis, and also was able to achieve lung concentrations above the required IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation that overcomes niclosamide’s limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

In drug dependence studies, rats are often tested daily with short breaks (such as weekends) spent untested in their home cages. Research on alcohol models has suggested that breaks from continuous testing can transiently enhance self-administration (termed the “alcohol deprivation effect”). The present study explored whether the salience of cocaine-access cues is increased after skipping weekend cocaine and cue exposures. Ultrasonic vocalizations (USVs) of the 50-kHz class are emitted by rats exposed to intravenous cocaine and have been shown to increase with repeated drug exposure at the same dose level (sensitization). The present study found that over the course of several weeks of cocaine self- or yoked-administration pre-drug cues signaling forthcoming access or delivery of cocaine elicited marked amounts of anticipatory 50-kHz USVs, and that weekend deprivation from cues and cocaine exaggerated further the level of calling (more calls on Mondays compared to Fridays). Anticipatory USVs extinguished less rapidly when weekend access to unreinforced cues was denied. The results may have clinical implications, in that intermittently avoiding cues or context may enhance drug cue salience and resistance to extinction.

Ultrasonic vocalization (USV) in the 50-kHz range occurs in rats immediately upon first-time exposure to cocaine or amphetamine, and rapidly increases with repetitive drug exposure at the same dose. This sensitized positive-affect response to these drugs of abuse is persistent in that the peak level of USVs again appears when the drug is reintroduced after several weeks of drug discontinuation. The present study explored whether with enough experience USVs might be elicited, and gradually escalate, in anticipation of impending drug delivery. Rats were trained to self-administer (SA) cocaine intravenously by lever pressing 5 days per week for 4 weeks. Yoked rats received experimenter-delivered cocaine matching that of SA rats. USVs and locomotor activity were recorded during each 10-min period prior to 60-min drug access sessions. Extinction trials in which drug access was denied were then carried out over an additional 4-week period. After about a week of cocaine experience, both the SA and Yoked groups began to progressively increase USVs when placed in an environment that predicted forthcoming drug exposure. Extinction of anticipatory calls and locomotion occurred over days after drug access ended. USVs may be a useful model for specifically investigating the neural basis of drug anticipation and aid in developing and assessing new addiction treatment strategies for reducing craving and relapse.

Short 50-kilohertz (kHz) range frequency-modulated ultrasonic vocalizations (USVs) produced by rats and mice are unconditionally elicited by drugs of abuse or electrical stimulation that increase dopamine activity in the nucleus accumbens, and it has been suggested that they reflect “positive affect” or incentive motivational states associated with appetitive behavior. The repeated administration of amphetamine is known to not only produce “psychomotor” sensitization, but also to facilitate a number of appetitive behaviors, including conditioned drug pursuit behavior. We were interested, therefore, in whether amphetamine-induced 50-kHz USVs would also increase with repeated drug exposure. USV recordings were made during 5-min sessions immediately after a saline infusion, and again 4-5 hours later, after 1.0 mg/kg intravenous amphetamine exposure. These sessions took place every other day over a 5-day period. A challenge dose of 1.0 mg/kg amphetamine was administered 2 weeks later to determine whether sensitization would persist. The initial amphetamine infusion increased 50-kHz USVs relative to the saline infusion. This effect was enhanced over trials and during the amphetamine challenge two weeks later. Classification of 50-kHz range call types revealed that complex frequency-modulated trill calls were sensitized by amphetamine, but not flat 50-kHz calls. It is possible that 50-kHz USV recordings could provide a potentially valuable behavioral measure of sensitization linked to enhanced incentive salience and increased tendency to self-administer drugs of abuse.

Cocaine is known to enhance nucleus accumbens dopamine (NAcc DA), to serve as a positive reinforcer and to produce negative effects, such as anxiety. The influence of diazepam on cocaine intake, cocaine-stimulated behavioral activity and NAcc DA was investigated using self-administration and experimenter-administered intravenous (i.v.) cocaine. In Experiment 1, rats were pretreated with diazepam (0.25 mg/kg) or saline (0.1 ml) 30 min prior to 20 daily 1-hour cocaine (0.75 mg/kg/injection) self-administration sessions. Cocaine intake increased for all animals across sessions, but was highest in diazepam-pretreated animals. Diazepam rats also self-administered their first cocaine injection of each session faster than controls. Experiment 2 utilized in vivo microdialysis to assess NAcc DA levels before and after experimenter-administered i.v. cocaine injections (0.75 mg/kg/injection x 2; 10-min interval) in diazepam- and saline-pretreated rats. Group differences were not revealed across basal and cocaine-stimulated NAcc DA assessments, indicating that diazepam did not decrease NAcc DA during cocaine self-administration. Findings that diazepam enhances cocaine self-administration and decreases cocaine response latency support the notion that cocaine-induced anxiety limits voluntary cocaine intake. It is further suggested that individual variations in cocaine-induced aversive effects may determine whether cocaine use is avoided or repeated.

The nucleus accumbens (NAc) is a central component of the mesocorticolimbic reward system. Increasing evidence strongly implicates long-term synaptic neuroadaptations in glutamatergic excitatory activity of the NAc shell and/or core medium spiny neurons in response to chronic drug and alcohol exposure. Such neuroadaptations likely play a critical role in the development and expression of drug-seeking behaviors. We have observed unique cell-type-specific bidirectional changes in NAc synaptic plasticity (metaplasticity) following acute and chronic intermittent ethanol exposure. Other investigators have also previously observed similar metaplasticity in the NAc following exposure to psychostimulants, opiates, and amazingly, even following an anhedonia-inducing experience. Considering that the proteome of the postsynaptic density likely contains hundreds of biochemicals, proteins and other components and regulators, we believe that there is a large number of potential molecular sites through which accumbal metaplasticity may be involved in chronic alcohol abuse. Many of our companion laboratories are now engaged in identifying and screening medications targeting candidate genes and its products previously linked to maladaptive alcohol phenotypes. We hypothesize that if manipulation of such target genes and their products change NAc plasticity, then that observation constitutes an important validation step for the development of novel therapeutics to treat alcohol dependence.

Adult rats often produce 50-kHz ultrasonic vocalizations (USVs), particularly the frequency-modulated varieties, in appetitive situations. These calls are thought by some to reflect positive affective states and the reinforcing value of drugs such as amphetamine and cocaine. The objective of this study was to determine whether the number of unconditioned 50-kHz USVs elicited by amphetamine predicts the development and/or magnitude of drug-conditioned motivation. In three experiments, we recorded USVs before and after injections of 1 mg/kg amphetamine (i.v. or i.p.) administered once per session. Rats were categorized as “high callers” or “low callers” according to individual differences in the number of 50-kHz USVs elicited by their first amphetamine injection. We examined the conditioned appetitive behavior and conditioned place preference (CPP) that emerged in high and low callers after repeated pairings of amphetamine with specific contexts. We also examined whether amphetamine-induced calling was affected by treatment within an unfamiliar (test chamber) versus familiar (home cage) context. Within an unfamiliar environment, the high callers consistently produced more amphetamine-induced 50-kHz USVs than the low callers. Compared to the low callers, high callers showed significantly greater amphetamine CPP as well as enhanced conditioned 50-kHz USVs and locomotor activity during anticipation of amphetamine. Individual differences were stable when amphetamine was administered in test chambers, but when it was administered in home cages, low callers showed an increase in 50-kHz calling that matched the high callers. These findings suggest that individual differences in drug-induced USVs can reveal environment-sensitive traits involved in drug-related appetitive motivation.

Subtypes of 50-kHz ultrasonic vocalizations (USVs) in rats are thought to reflect positive affect and occur with cocaine or amphetamine delivery. In contexts predicting forthcoming cocaine, pre-drug anticipatory USVs are initially minimal during daily sessions but gradually escalate over several weeks, presumably as the animal learns to expect and look forward to impending drug access. To gain more insight into motivational aspects of cocaine intake in animal models of drug dependence studies, it is important to compare experience-dependent changes in lever response rate, USVs, and locomotion during cocaine conditioning and extinction trials. To address whether cocaine-induced increases in lever responding and locomotor activity correspond with USV production. The study also determined whether short-term cocaine and context deprivation effects could be detected during conditioning or extinction. Rats underwent 20 days of 60-min sessions of self- or yoked administration of cocaine (0.75 mg/kg/infusion, i.v.), followed by 19 days of extinction training (8 weeks total, weekends off). Lever responding for cocaine and cocaine-induced locomotor activity increased across conditioning sessions. In contrast, the number of frequency modulated 50-kHz USVs evoked in response to cocaine infusion decreased with cocaine experience, suggesting perhaps tolerance to the rewarding properties of the drug. In addition, USVs but not lever pressing or locomotion are affected after brief periods of drug and/or drug context abstinence. Except for initial drug exposure, increased cocaine seeking during cocaine delivery could reflect either enhanced drug motivation or the development of drug tolerance, but not enhanced positive affect.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Hyperthermia and hyperlocomotor activity are commonly reported acute effects of high dose, experimenter-delivered 3,4-methylenedioxymethamphetamine (MDMA). The current investigation was performed to determine short- to long-term physiological and behavioral changes induced by moderate intake MDMA self-administration. In the present study, rats self-administered MDMA (approx. 2.0-7.0 mg/kg/day) across 20 days during daily 2-h operant sessions. Locomotor activity was assessed during MDMA self-administration sessions and core temperatures were recorded before and after each session. Findings of the first several sessions showed core temperatures significantly decreased after MDMA self-administration compared to baseline and to a control group that self-administered saline during operant sessions. As sessions proceeded, the MDMA-induced hypothermic response diminished and core temperatures normalized, then increased during the last few sessions. Also, locomotor activity during MDMA self-administration sessions was initially equivalent to saline level activity, but increased by day 8 to significantly greater levels. Our findings demonstrate experience-dependent changes after voluntary administration of MDMA that are clearly observable in temperature regulation and behavioral activity.