A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here we characterize a novel pan-RAS inhibitor, ADT-007, that potently and selectively inhibited the growth of histologically diverse cancer cell lines with mutant or activated RAS irrespective of the RAS mutation or isozyme. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-007 bound RAS in lysates from sensitive cells with sub-nanomolar EC 50 values but did not bind RAS in lysates from insensitive cells with low activated RAS. Insensitivity to ADT-007 was attributed to metabolic deactivation by UGT-mediated glucuronidation, providing a detoxification mechanism to protect normal cells from pan-RAS inhibition. Molecular modeling and experiments using recombinant RAS revealed that ADT-007 binds RAS in a nucleotide-free conformation to block GTP activation. Local injection of ADT-007 strongly inhibited tumor growth in syngeneic immune competent and xenogeneic immune deficient mouse models of colorectal and pancreatic cancer and activated innate and adaptive immunity in the tumor microenvironment. SIGNIFICANCE: ADT-007 is a novel pan-RAS inhibitor with a unique mechanism of action having potential to circumvent resistance to mutant-specific KRAS inhibitors and activate antitumor immunity. The findings support further development of ADT-007 analogs and/or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy for RAS mutant cancers. BACKGROUND: It is projected that colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA) will cause 52,580 and 49,830 deaths in the US in 2023, respectively (1). The 5-year survival rates for CRC and PDA are 65% and 12%, respectively (1). Over 50% of CRC and 90% of PDA patients harbor mutations in KRAS genes that are associated with poor prognosis, making the development of novel KRAS inhibitors an urgent unmet medical need (2).

The Active-Site [4Fe-4S] Cluster in the Isoprenoid Biosynthesis Enzyme IspH Adopts Unexpected Redox States during Ligand Binding and Catalysis.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S]2+ cluster. Reduction generates [4Fe-4S]+ (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S]+ species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

Fracking pond restoration in the western Rio Grande Plains, Texas: Stockpiled topsoils may not be necessary, but seeding helps.

Soil disturbances that accompany energy development can damage local habitats. Prior to oil and gas extraction, it is commonly recommended that topsoil stockpiles be created to aid future restoration. Our study area, a retired fracking pond in the western Rio Grande Plains, Texas, was restored in 2017 with stockpiled topsoil that was collected in 2013. We segregated the existing stockpile into three layers that were ∼1.5 m in thickness and distributed these layers, along with a non-amended control surface (consisting of former subsoil that made up the perimeter of the fracking pond), in strips over the restoration area. Each of the four surfaces was seeded with a mixture of (1) 13 native grasses, (2) 13 native grasses plus an annual warm-season grass cover crop, or (3) non-seeded. We monitored plant density and species composition two through five years post-restoration. The non-amended control surface had higher seeded grass density during the final 2 sampling periods; stockpiled surfaces seldom differed from each other. Previous year's competing plant density had little effect on restoration success. Providing supplemental seed initially increased seeded plant density but benefits diminished over time; adding a cover crop was not advantageous. Changes in community composition over time were similar on stockpile surfaces but more variable than observed on the control surface. Results suggest that stockpiling topsoil may not be necessary, but that supplemental seeding was beneficial, to restoration success.

Provider survey of the roles of clinical pharmacists in primary care in a Federally Qualified Health Center versus an Accountable Care Organization.

Background: Healthcare providers at primary care Federally Qualified Healthcare Centers (FQHC) and an Accountable Care Organizations (ACO) collaborated with clinical pharmacists in providing patient care utilizing the comprehensive medication management (CMM) framework. The intention of CMM was to generate more time for providers to see patients, and to improve overall patient quality of life. Objectives: The purpose of this study was to survey the providers' views of clinical pharmacy services, and to compare and contrast the shared-visit model in rural FQHCs and an ACO collaborative practice agreement model in a mid-sized metropolitan area. Methods: Primary care providers completed a five-domain 22-item survey of provider patient care, provider pharmacy consults, provider ranking of pharmacy-services, disease treatment and provider views on the value of clinical pharmacists. Results: FQHC pharmacists were available one day per week (75%), while 69% of ACO pharmacists were available five days per week. FQHC providers requested <5 pharmacist consults per week (46%), while ACO requested >10 consults per week (44%). Both organizations had nearly identical provider rankings and impact on patient care for clinical pharmacy services and disease-focused pharmacy services. The provider survey of satisfaction with pharmacy consultations were highly positive and scored as strongly agree with FQHC and ACO, with the exception of three items with the FQHC. Overall providers at both organizations report highly effective medication-related improvements, disease outcomes and also recommend clinical pharmacists to other providers and primary care teams. Regression analysis revealed relevant clinical associations between survey statements not seen with individual survey items alone. Conclusion: Primary care providers report high satisfaction with, and benefits of, clinical pharmacy services. Drug information resource and disease-focused management were documented by providers as valuable pharmacy services. Providers promoted expanding the role of clinical pharmacists with providers, and integration into primary care teams.

Hispolon Cyclodextrin Complexes and Their Inclusion in Liposomes for Enhanced Delivery in Melanoma Cell Lines.

Hispolon, a phenolic pigment isolated from the mushroom species Phellinus linteus, has been investigated for anti-inflammatory, antioxidant, and anticancer properties; however, low solubility and poor bioavailability have limited its potential clinical translation. In this study, the inclusion complex of hispolon with Sulfobutylether-ß-cyclodextrin (SBEßCD) was characterized, and the Hispolon-SBEßCD Complex (HSC) was included within the sterically stabilized liposomes (SL) to further investigate its anticancer activity against melanoma cell lines. The HSC-trapped-Liposome (HSC-SL) formulation was investigated for its sustained drug delivery and enhanced cytotoxicity. The inclusion complex in the solid=state was confirmed by a Job's plot analysis, molecular modeling, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), Proton nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). The HSC-SL showed no appreciable deviation in size (<150 nm) and polydispersity index (<0.2) and improved drug encapsulation efficiency (>90%) as compared to control hispolon liposomes. Individually incorporated hispolon and SBEßCD in the liposomes (H-CD-SL) was not significant in loading the drug in the liposomes, compared to HSC-SL, as a substantial amount of free drug was separated during dialysis. The HSC-SL formulation showed a sustained release compared to hispolon liposomes (H-SLs) and Hispolon-SBEßCD liposomes (H-CD-SLs). The anticancer activity on melanoma cell lines (B16BL6) of HSC and HSC-SL was higher than in H-CD-SL and hispolon solution. These findings suggest that HSC inclusion in the HSC-SL liposomes stands out as a potential formulation approach for enhancing drug loading, encapsulation, and chemotherapeutic efficiency of hispolon and similar water insoluble drug molecules.

Treatment-Associated Acute Lymphoblastic Leukemia Following Autologous Hematopoietic Stem Cell Transplant and Lenalidomide Maintenance in Patients With Multiple Myeloma.

Secondary malignancies including leukemia are an increasing concern in patients with prior primary malignancies treated with alkylating agents or topoisomerase II inhibitors. These can also be referred to as therapy-related leukemia. Therapy-related leukemia most commonly results in myelodysplastic syndrome or acute myeloid leukemia. The alkylating agent can cause chromosomal aberrations typically manifest as deletions in chromosome 11 or loss of part of complete loss of chromosomes 5 and 7. Conversely, acute lymphoblastic leukemia (ALL) has been described following maintenance therapy with immunomodulatory (IMiD) drugs pomalidomide, thalidomide, and lenalidomide. We present a case of a 71-year-old man with a history of multiple myeloma (MM) maintained on lenalidomide after stem cell transplant who presented with treatment-associated ALL. At time of leukemic presentation, chromosomal analysis showed a near-triploid clone consistent with masked double low hyplodiploidy which is associated with a poor prognosis. The patient had a deletion of the long arm of chromosome 5 which has been described in prior case reports with ALL secondary to lenalidomide therapy. There are explicit mechanisms in the literature, which have been attributed to development of ALL after exposure to thalidomide or lenalidomide. At time of submission, there are 20 cases described in the literature linking ALL to IMiD drugs. We describe a case and review the mechanisms of lenalidomide-associated ALL.

Secondary Extramedullary Myeloma of the Gallbladder: A Case Report.

Extramedullary myeloma (EMM) is an infrequent but well-established manifestation of multiple myeloma (MM), defined as a soft tissue plasma cell neoplasm without bone marrow involvement. Gallbladder involvement in EMM, however, is a very rare occurrence, with only 8 cases found in the English medical literature. Here, we present a case of an older adult male with a gallbladder mass in the presence of increasing serum kappa light chains after a normal bone marrow biopsy confirmed the complete remission of a previous MM diagnosis. Histopathologic evaluation of a biopsied sample confirmed the mass as an atypical plasma cell neoplasm. Later in his treatment, he developed several firm, smooth, violaceous skin nodules on the torso, which histopathology confirmed as also being atypical plasma cell neoplasms. We aim to contribute to the medical literature by expanding the pool of information regarding EMM of the gallbladder to support future diagnostic and treatment recommendations.

Hepatitis B Virus Reactivation Following Treatment of HNSCC With Cisplatin.

Hepatitis B Virus (HBV) reactivation is a known complication of intense immunosuppression with B-cell depleting monoclonal antibody therapy and transplantation immunosuppression. HBV reactivation has occurred following treatment with chemotherapy regimens for hematologic malignancies and solid tumors. There are 2 prior case reports of HBV reactivation following cisplatin monotherapy for head and neck squamous cell carcinoma (HNSCC). Here, we present a case of a 49-year-old Caucasian male with a past medical history of laryngeal squamous cell carcinoma (SCC). There are no consensus guidelines on how to define hepatitis B reactivation. There are guidelines on when to initiate prophylaxis with Entecavir while on immunosuppressive therapy with risk according to medication category and hepatitis B surface antigen/hepatitis B core antibody IgG serology. CDC recommends screening everyone. American Society of Clinical Oncology (ASCO) now with a recent update in 2020 recommends screening everyone. There is a definite role of immunosuppression in HBV reactivation, however, there is also direct enhancement by cisplatin of viral replication by creating endoplasmic reticulum stress which increases HBV DNA indirectly. Finally, cytotoxicity enhances HBV reactivation and immune reconstitution post withdrawing immunosuppressive treatment. Because of the effects of chemotherapy, aka cisplatin goes beyond immunosuppression-related reactivation of HBV, our recommendations are in line with CDC and ASCO to screen all patients for HBV before onset of chemotherapy and start Entecavir/Tenofovir Disoproxil Fumarate before the onset of chemotherapy for HBV-positive patients.

Difluprednate-Hydroxypropyl-ß-Cyclodextrin-Based Ophthalmic Solution for Improved Delivery in a Porcine Eye Model.

Purpose: Difluprednate (DFP) is an approved corticosteroid, available as an ophthalmic emulsion (Durezol®), used to treat pain and inflammation of the eye following ocular surgeries. This study utilized hydroxypropyl-ß-cyclodextrin (HPBCD)-based DFP ophthalmic solution for improved ocular delivery. Methods: The DFP-HPBCD complex formation was studied in the liquid and solid states. Phase solubility, molecular docking studies, differential scanning calorimetry, and Fourier transform infrared spectroscopy suggested inclusion complexation of DFP and HPBCD. Results: DFP-HPBCD-based eye drops (solution) provided 16 and 26 times higher transcorneal permeation when compared to the suspension (no HPBCD, control) and Durezol, respectively (P < 0.001). In addition, ocular drug distribution studies conducted in continuously perfused whole porcine eyes showed DFP permeated into all of the ocular tissues in significantly higher amounts than Durezol. Conclusions: The solution-based eye drops in this study is iso-osmotic, safe, and more permeable in porcine eyes compared to Durezol.

Cardioprotective Effects of Oroxylum indicum Extract Against Doxorubicin and Cyclophosphamide-Induced Cardiotoxicity.

Administration of Chemotherapeutics, especially doxorubicin (DOX) and cyclophosphamide (CPS), is commonly associated with adverse effects such as myelosuppression and cardiotoxicity. At this time, few approved therapeutic options are currently available for the management of chemotherapy-associated cardiotoxicity. Thus, identification of novel therapeutics with potent cardioprotective properties and minimal adverse effects are pertinent in treating Doxorubicin and Cyclophosphamide-induced cardiotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) is a natural product known to possess several beneficial biological functions including antioxidant, anti-inflammatory and cytoprotective effects. We therefore set to investigate the cardioprotective effects of OIE against Doxorubicin and Cyclophosphamide-induced cardiotoxicity and explore the potential cardioprotective mechanisms involved. Adult male mice were treated with DOX and CPS in combination, OIE alone, or a combination of OIE and DOX & CPS. Swimming test was performed to assess cardiac function. Markers of oxidative stress were assessed by levels of reactive oxygen species (ROS), nitrite, hydrogen peroxide, catalase, and glutathione content. The activity of interleukin converting enzyme and cyclooxygenase was determined as markers of inflammation. Mitochondrial function was assessed by measuring Complex-I activity. Apoptosis was assessed by Caspase-3 and protease activity. Mice treated with DOX and CPS exhibited reduced swim rate, increased oxidative stress, increased inflammation, and apoptosis in the heart tissue. These cardiotoxic effects were significantly reduced by co-administration of OIE. Furthermore, computational molecular docking studies revealed potential binding of DOX and CPS to tyrosine hydroxylase which validated our in vivo findings regarding the inhibition of tyrosine hydroxylase activity. Our current findings indicated that OIE counteracts Doxorubicin and Cyclophosphamide-induced cardiotoxicity-through inhibition of ROS-mediated apoptosis and by blocking the effect on tyrosine hydroxylase. Taken together, our findings suggested that OIE possesses cardioprotective effects to counteract potentially fatal cardiac complications associated with chemotherapy treatment.

Emerging SARS-CoV-2 Variants: A Review of Its Mutations, Its Implications and Vaccine Efficacy.

The widespread increase in multiple severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants is causing a significant health concern in the United States and worldwide. These variants exhibit increased transmissibility, cause more severe disease, exhibit evasive immune properties, impair neutralization by antibodies from vaccinated individuals or convalescence sera, and reinfection. The Centers for Disease Control and Prevention (CDC) has classified SARS-CoV-2 variants into variants of interest, variants of concern, and variants of high consequence. Currently, four variants of concern (B.1.1.7, B.1.351, P.1, and B.1.617.2) and several variants of interests (B.1.526, B.1.525, and P.2) are characterized and are essential for close monitoring. In this review, we discuss the different SARS-CoV-2 variants, emphasizing variants of concern circulating the world and highlight the various mutations and how these mutations affect the characteristics of the virus. In addition, we discuss the most common vaccines and the various studies concerning the efficacy of these vaccines against different variants of concern.

ß-hydroxybutyric acid attenuates oxidative stress and improves markers of mitochondrial function in the HT-22 hippocampal cell line.

Ketone bodies have been the topic of research for their possible therapeutic neurotropic effects in various neurological diseases such as Parkinson's disease, dementia, and seizures. However, continuing research on ketone bodies as a prophylactic agent for decreasing the risk for various neurodegenerative diseases is currently required. In this paper, hippocampal HT-22 cells were treated with ß-hydroxybutyric acid at different doses to elucidate the neurotropic effects. In addition, markers of oxidative stress, mitochondrial function, and apoptosis were investigated. As a result, the ketone body (ß-hydroxybutyric acid) showed a significant increase in hippocampal neuronal viability at a moderate dose. Results show that ß-hydroxybutyric acid exhibited antioxidant effect by decreasing prooxidant oxidative stress markers such as reactive oxygen species, nitrite content, and increasing glutathione content leading to decreased lipid peroxidation. Results show that ß-hydroxybutyric acid improved mitochondrial functions by increasing Complex-I and Complex-IV activities and showing that ß-hydroxybutyric acid significantly reduces caspase-1 and caspase-3 activities. Finally, using computational pharmacokinetics and molecular modeling software, we validated the pharmacokinetic effects and pharmacodynamic (N-Methyl-D-aspartic acid and acetylcholinesterase) interactions of ß-hydroxybutyric acid. The computational studies demonstrate that ß-hydroxybutyric acid can interact with N-Methyl-D-aspartic acid receptor and cholinesterase enzyme (the prime pharmacodynamic targets for cognitive impairment) and further validates its oral absorption, distribution into the central nervous system. Therefore, this work highlights the neuroprotective potential of ketone bodies in cognitive-related neurodegenerative diseases.

Nutraceutical based SIRT3 activators as therapeutic targets in Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and its incidence is increasing worldwide with increased lifespan. Currently, there is no effective treatment to cure or prevent the progression of AD, which indicates the need to develop novel therapeutic targets and agents. Sirtuins, especially SIRT3, a mitochondrial deacetylase, are NAD-dependent histone deacetylases involved in aging and longevity. Accumulating evidence indicates that SIRT3 dysfunction is strongly associated with pathologies of AD, hence, therapeutic modulation of SIRT3 activity may be a novel application to ameliorate the pathologies of AD. Natural products commonly used in traditional medicine have wide utility and appear to have therapeutic benefits for the treatment of neurodegenerative diseases such as AD. The present review summarizes the currently available natural SIRT3 activators and their potentially neuroprotective molecular mechanisms of action that make them a promising agent in the treatment and management of neurodegenerative diseases such as AD.

Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics.

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

Enhanced Bioavailability of Boswellic Acid by Piper longum: A Computational and Pharmacokinetic Study.

Chronic inflammation is a key culprit factor in the onset and progression of several diseases. Novel and pharmacologically effective therapeutic approaches are needed for new treatment remedy or improved pharmacokinetics and pharmacodynamics for existing synthetic drugs, in particular natural products. Boswellic acids are well-known natural products, with capacity to effectively retard inflammation without severe adverse effects. However, the therapeutic use of Boswellic acids are greatly hindered by its poor pharmacokinetic properties. Co-administration strategies that facilitate the oral absorption and distribution of Boswellic acids should lead to a safe and more effective use of this product prophylactically and therapeutically in inflammatory disorders. In this study, we examined the effect of Piper longum extract on the absorption and bioavailability of Boswellic acid in rabbits. In addition, we further explored computational pharmacodynamic interactions between Piper longum and Boswellic acid. Piper longum extract at 2.5 and 10 mg/kg, increased the bioavailability of Boswellic acid (p < 0.05). Based on our drug-based computational modeling, cytochrome P450 (CYP450)-mediated mechanism was involved in increased bioavailability. These findings confirmed that Piper longum with Boswellic acid may be administered orally together for effective therapeutic efficacy. Thus, our studies support the application of Piper longum with Boswellic acid as a novel therapeutic avenue in diseases associated with inflammation.

Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Infrared (GC-IR) Analyses of the Chloro-1- n-pentyl-3-(1-naphthoyl)-Indoles: Regioisomeric Cannabinoids.

The analytical differentiation of the indole ring regioisomeric chloro-1- n-pentyl-3-(1-naphthoyl)-indoles is described in this report. The regioisomeric chloroindole precursor compounds, N- n-pentyl chloroindole synthetic intermediates, and the target chloro-substituted naphthoylindoles showed the equivalent gas chromatographic elution order based on the position of chlorine substitution on the indole ring. The regioisomeric chloro-1- n-pentyl-3-(1-naphthoyl)-indoles yield electron ionization mass spectra having equivalent major fragments resulting from cleavage of the groups attached to the central indole nucleus. Fragment ions occur at m/z 127 and 155 for the naphthyl and naphthoyl cations common to all indoles having the naphthoyl group substituted at the indole-3 position. Fragments resulting from the loss of the naphthoyl and/or n-pentyl groups from the molecular radical cation yield the cations at m/z 318, 304, 248, and 178. The characteristic (M-17)+ fragment ion at m/z 358 resulting from the loss of OH radical is significant in the mass spectra of all these compounds with 1-naphthoyl groups substituted at the indole-3 position. The vapor phase infrared spectra provide a number of characteristic absorption bands to identify the individual isomers.

Improved Ocular Delivery of Nepafenac by Cyclodextrin Complexation.

Nepafenac is a nonsteroidal anti-inflammatory drug (NSAID), currently only available as 0.1% ophthalmic suspension (Nevanac®). This study utilized hydroxypropyl-ß-cyclodextrin (HPBCD) to increase the water solubility and trans-corneal permeation of nepafenac. The nepafenac-HPBCD complexation in the liquid and solid states were confirmed by phase solubility, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR) analyses. Nepafenac 0.1% ophthalmic solution was formulated using HPBCD (same pH and osmolality as that of Nevanac®) and pig eye trans-corneal permeation was studied versus Nevanac®. Furthermore, nepafenac content in cornea, sclera, iris, lens, aqueous humor, choroid, ciliary body, retina, and vitreous humor was studied in a continuous isolated pig eye perfusion model in comparison to the suspension and Nevanac®. Permeation studies using porcine corneas revealed that the solution formulation had a permeation rate 18 times higher than Nevanac®. Furthermore, the solution had 11 times higher corneal retention than Nevanac®. Drug distribution studies using porcine eyes revealed that the solution formulation enables detectable levels in various ocular tissues while the drug was undetectable by Nevanac®. The ocular solution formulation had a significantly higher drug concentration in the cornea compared to the suspension or Nevanac®.

GC-MS and GC-IR Analyses of the Methoxy-1-n-pentyl-3-(1-naphthoyl)-indoles: Regioisomeric Designer Cannabinoids.

The indole ring regioisomeric methoxy-1-n-pentyl-3-(1-naphthoyl)-indoles represent indole ring-substituted analogs of the synthetic cannabinoid JWH-018. The electron ionization mass spectra show equivalent regioisomeric major fragments resulting from cleavage of the groups attached to the central indole nucleus. The characteristic (M-17)+ fragment ion at m/z 354 resulting from the loss of OH group is significant in the mass spectra of all four compounds. Fragmentation of the naphthoyl and/or pentyl groups yields the cations at m/z 314, 300, 244 and 216. The vapor-phase infrared spectra provide a number of characteristic absorption bands to identify the individual isomers. Gas chromatographic separations on a capillary column containing a film of trifluoropropylmethyl polysiloxane (Rtx-200) provided excellent resolution of these compounds, their precursor indoles and intermediate pentylindoles. The elution order appears related to the degree of crowding of indole ring substituents.

Analytical studies on the 2-naphthoyl substituted-1-n-pentylindoles: Regioisomeric synthetic cannabinoids.

The six 1-n-pentyl-2-, 3-, 4-, 5-, 6- and 7-(2-naphthoyl)-indoles each have the same substituents attached to the indole ring, identical elemental composition (C24H23NO) yielding identical nominal and accurate masses. The electron ionization mass spectra of the 2-naphthoyl substituted isomers share equivalent major fragment ions resulting from cleavage of the groups attached to the central indole nucleus with some differences in relative abundances. These six regioisomers were successfully resolved on an Rtx-5 and Rxi-17Sil MS stationary phases and the molecules having both substituent groups on the same side of the indole ring (1,2- and 1,7-substituents) show the least retention. The more linear molecules have higher relative retention properties. A comparison of the GC properties of the 1-naphthoyl- and 2-naphthoyl groups attached at identical positions of the indole ring showed higher GC retention for the 2-naphthoyl substituted isomer in all cases evaluated. The amide inverse isomers (1-naphthoyl-3-n-pentylindoles) were separated from the 1-n-pentyl-3-naphthoyl-indoles on an Rtx-200 stationary phase. The two inverse amide isomers having the 1- and 2-naphthoyl groups substituted at the 1-position of the indole ring elute before either of the N-alkyl-indole isomers having the 1- and 2-naphthoyl groups substituted at the 3-position of the indole ring. The amide inverse isomers yield EI mass spectra easily distinguishing these amides from the ketone isomers having the naphthoyl groups at the indole 3-position.

Correlation of vapor phase infrared spectra and regioisomeric structure in synthetic cannabinoids.

The twelve 1-n-pentyl-2-, 3-, 4-, 5-, 6- and 7-(1- and 2-naphthoyl)-indoles each have the same substituents attached to the indole ring, identical elemental composition (C24H23NO) yielding identical nominal and accurate masses. These twelve isomers cover all possible positions of carbonyl bridge substitution for both indole (positons 2-7) and naphthalene rings (positions 1 and 2). Regioisomeric compounds can represent significant challenges for mass based analytical methods however, infrared spectroscopy is a powerful tool for the identification of positional isomers in organic compounds. The vapor phase infrared spectra of these twelve uniquely similar compounds were evaluated in GC-IR experiments. These spectra show the bridge position on the indole ring is a dominating influence over the carbonyl absorption frequency observed for these compounds. Substitution on the pyrrole moiety of the indole ring yields the lowest CO frequency values for position 2 and 3 giving a narrow range from 1656 to 1654cm-1. Carbonyl absorption frequencies are higher when the naphthoyl group is attached to the benzene portion of the indole ring yielding absorption values from 1674 to 1671cm-1. The aliphatic stretching bands in the 2900cm-1 region yield a consistent triplet pattern because the N-alkyl substituent tail group remains unchanged for all twelve regioisomers. The asymmetric CH2 stretch is the most intense of these three bands. Changes in positional bonding for both the indole and naphthalene ring systems results in unique patterns within the 700 wavenumber out-of-plane region and these absorption bands are different for all 12 regioisomers.