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The prevalence of cannabis use has been increasing among both adolescents and adults worldwide. New trends in cannabis legalization and enhanced social media marketing have led to the availability of multiple high-potency cannabis products with hundreds of new and powerful delivery systems. Over the last decade, there have been drastic changes in cannabis formulations, potency, routes of consumption, and device technology, with increased complexity and sophistication among growers, suppliers, and consumers. Patterns of cannabis use among patients can have important clinical implications, including acute neurocognitive effects, chronic multiorgan toxicity, psychiatric, behavioral, social, and economic impact. However, assessment of medical or surgical patients who use cannabis either recreationally or problematically has become challenging for the clinician due to the changing patterns of cannabis consumption. This review provides information on the clinical evaluation of patients who use cannabis in a problematic fashion, with the focus on tetrahydrocannabinol. It provides the clinician with knowledge regarding cannabis terminology, sources, pharmacology, routes of administration, formulations, dosing, and toxicities. Using these components, an assessment approach for diagnosing cannabis use disorder is synthesized at the conclusion of the article., Competing Interests: Conflicts of Interest None declared

Background: Breakthrough seizures and status epilepticus require urgent management. Administration of intravenous push (IVP) levetiracetam has been demonstrated to be safe as compared to intravenous piggyback (IVPB). This transition can potentially offer faster time to administration and reduced drug and material cost. The objective of this study was to observe safety of administration in patients receiving levetiracetam via IVP compared to IVPB in acute care settings. Methods: This is a multi-center, observational, retrospective cohort study of 1214 adult patients who received levetiracetam pre- and post-implementation of IVP over a 6 month timespan. Primary outcome was time from order verification to administration of urgent first-time doses. Secondary outcomes included time to administration of loading doses and cost. Safety outcome was infusion site related reactions. Results: Time from order verification to administration of urgent first-time doses pre- and post-implementation of IVP administration was reduced from 61 minutes to 47 minutes ( P =0.0002). Infusion site related reactions were observed in 6 out of 5432 doses in the IVPB arm and in 5 out of 4700 doses in the IVP arm ( P =1). Total estimated cost was $76,171.96 for the 5449 IVPB total doses and $11,484.33 for the 4721 IVP total doses. Conclusions: Transition from IVPB to IVP administration reduced time from order verification to administration of urgent first-time doses with both administrations having similar incidence of infusion site related reactions. Cost savings and improved workflow were observed. Levetiracetam administered via IVP may be considered as a safe alternative method of administration in the acute care setting., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Enoxaparin is a hydrophilic drug with obesity having little effect on its apparent volume of distribution, therefore patients with obesity receiving standard 1 mg/kg dosing may be at a higher risk of supratherapeutic dosing. Conversely, dose reducing patients with obesity could place already at risk patients at higher risk of a thrombotic event. Data and recommendations are variable for the most appropriate weight-based dose of therapeutic enoxaparin in obese patients, particularly those a weight > 100 kg or a body mass index (BMI) ≥ 40 kg/m 2 . The purpose of this systematic review was to globally evaluate these data to surmise optimal dosing recommendations for patients with obesity. A systematic review of English language studies was conducted and identified articles via Pubmed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) searches. Studies were included if they reported therapeutic enoxaparin use in adult patients with a BMI ≥ 40 kg/m 2 or body weight > 100 kg and the percentage of patients achieving a therapeutic anti-Xa based on a weight-based dose or the weight-based dose required to produce a therapeutic anti-Xa level. Therapeutic attainment of anti-Xa levels were assessed across enoxaparin weight-based dosing categories including a very low dose group: < 0.75 mg/kg, low dose group: 0.75-0.85 mg/kg, and standard dose group: ≥ 0.95 mg/kg. Rates of bleeding and thrombosis were also evaluated. A total of eight studies were included. For anti-Xa level assessment, 682 patients were included. A total of 62% of anti-Xa levels were therapeutic in the very low dose group, 66% in the low dose group, and 42% in the standard dose group. Overall rates of total bleeding and thrombosis were assessed in 798 patients. A total of 29 bleedings (3.6%) occurred, and 27 reported a relationship to dose. Most bleedings, 85.2% (n = 23/27), occurred with doses in the standard dose group (≥ 0.95 mg/kg). Thrombosis occurred in 5 patients (0.6%). Utilization of a reduced weight-based dosing strategy for therapeutic enoxaparin in obese patients may increase the percentage of patients with a therapeutic anti-Xa level., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Although inhalant abuse is common, it is one of the most neglected and overlooked forms of substance abuse. Inhalants refer to a wide variety of substances including volatile solvents, aerosols, gases, and nitrites. The mechanism of action of inhalants has not been fully defined. Several molecular targets contribute to the pharmacology, including ion-channel proteins that control neuronal excitability. These agents interact with various receptors and can cause changes in cell-membrane fluidity and nerve-membrane ion channels. Three main pharmacologic categories of inhalants, namely, volatile solvents and anesthetic gases, nitrous oxide, and volatile alkyl nitrites, have distinct pharmacologies, mechanisms of action, and toxicities. Inhalants are linked to multisystem damage affecting the pulmonary, cardiac, dermatologic, renal, hematologic, gastrointestinal, hepatic, and neurologic systems. Chronic inhalant abuse can also cause psychiatric, cognitive, behavioral, and anatomical deficits in humans, leading to reduced productivity and quality of life. Inhalant abuse during pregnancy is associated with fetal abnormalities. Clinical assessment for inhalant abuse should be done systematically. After decontamination and stabilization of the patient, further history and physical examination is necessary to establish an appropriate diagnosis based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition . Laboratory testing for inhalant abuse is very limited, and imaging studies may be helpful in certain situations. The treatment of inhalant use disorder is similar to that of other substance abuse disorders and includes supportive care, pharmacotherapy, and behavioral therapy. Preventive measures are essential., Competing Interests: Conflicts of InterestNone declared

BACKGROUND Liposuction is a commonly performed aesthetic procedure. New technology is being integrated to target fine lines or creases in the skin, known as rhytides, and skin laxity that cannot be corrected with liposuction. "Liposculpture" is a new term to describe a variation of liposuction that integrates this new technology for both fat reduction and skin tightening. A new form of liposculpture known as Renuvion, which utilizes a helium-based plasma technology, is being added to improve cosmetic outcomes. In this case report, we describe a case of internal thermal injury masquerading as cellulitis from the use of this new technology. CASE REPORT A 37-year-old African-American woman with a history of anemia, hypertension, hyperlipidemia, and depression, with prior breast reduction and liposuction, presented to the emergency room with a 5-day history of waxing/waning fevers that began shortly after undergoing a liposculpture procedure. Imaging demonstrated subcutaneous emphysema and edema in the abdominal wall. Empiric antimicrobials were started given concern for surgical wound infection, but erythema and pain worsened despite treatment. Other infectious markers were negative including negative procalcitonin, normal white blood cell count, and no growth from wound and blood cultures, leading to consideration of a new diagnosis of thermal injury. Antibiotics were subsequently de-escalated to a combination of levofloxacin and doxycycline. She was also treated with topical silver sulfadiazine for her thermal injury. She required multiple rounds of infrared light therapy and lymphatic massage techniques with overall improvement but lasting hyperpigmentation by her 6-month follow-up. CONCLUSIONS Thermal injuries have been an extremely rare finding in patients undergoing cosmetic procedures. Treatments targeting skin laxity and rhytides may confer higher risk. It is important to note that presentations can mimic cellulitis or surgical site infection. In this case vignette, we describe a rare complication of thermal injury following a liposculpture procedure using a cold atmospheric plasma device in a previously healthy 37-year-old African-American woman.

Background: The 2018 Society of Critical Care Medicine guidelines on the "Prevention and Management of Pain, Agitation/Sedation, Delirium, Immobility, and Sleep Disruption in Adult Patients in the ICU" advocate for protocol-based analgosedation practices. There are limited data available to guide which analgesic to use. This study compares outcomes in patients who received continuous infusions of fentanyl or hydromorphone as sedative agents in the intensive care setting., Methods: This retrospective cohort study evaluated patients admitted into the medical intensive care unit, the surgical intensive care unit, and the cardiac intensive care unit from April 1, 2017, to August 1, 2018, who were placed on continuous analgesics. Patients were divided according to receipt of fentanyl or hydromorphone as a continuous infusion as a sedative agent. The primary endpoints were ICU length of stay and time on mechanical ventilation., Results: A total of 177 patients were included in the study; 103 received fentanyl as a continuous infusion, and 74 received hydromorphone as a continuous infusion. Baseline characteristics were similar between groups. Patients in the hydromorphone group had deeper sedation targets. Median ICU length of stay was eight days in the fentanyl group compared to seven days in the hydromorphone group (p = 0.11) and median time on mechanical ventilation was 146.47 hours in the fentanyl group and 122.33 hours in the hydromorphone group (p = 0.31). There were no statistically significant differences in the primary endpoints of ICU length of stay and time on mechanical ventilation between fentanyl and hydromorphone for analgosedation purposes., Conclusion: No statistically significant differences were found in the primary endpoints studied. Patients in the hydromorphone group required more tracheostomies, restraints, and were more likely to have a higher proportion of Critical Care Pain Observation Tool (CPOT) scores > 2., Competing Interests: Conflict of Interest Conflict of Interests: The author(s) declared no potential conflicts of interest concerning the research, authorship, and publication of this article., (© 2021 Hahnl Choi, Sara Radparvar, Samuel L. Aitken, Jerry Altshuler, published by Sciendo.)

Purpose: Nearly half of intensive care unit (ICU) patients will develop delirium. Antipsychotics are used routinely for the management of ICU delirium despite limited reliable data supporting this approach. The unwarranted continuation of antipsychotics initiated for ICU delirium is an emerging transitions of care concern, especially considering the adverse event profile of these agents. We sought to evaluate the magnitude of this issue across 6 centers in New Jersey and describe risk factors for continuation., Methods: This multicenter, retrospective study examined adult ICU patients who developed ICU delirium from June 2016 to June 2018. Patients were included in the study if they received at least 3 doses of antipsychotics while in the ICU with presence of either a clinical diagnosis of delirium or a positive Confusion Assessment Method score. Patients were excluded if they were on an antipsychotic before ICU admission., Results: Of the 300 patients included and initiated on antipsychotics for ICU delirium, 157 (52.3%) were continued on therapy upon transfer from the ICU to another level of inpatient care. The number of patients continued on newly initiated antipsychotics further increased to 183 (61%) upon discharge from the hospital., Conclusion: The continuation of antipsychotics for the management of delirium during transitions of care was a common practice across ICUs in New Jersey. Several risk factors for continuation of antipsychotics were identified. Efforts to reduce unnecessary continuation of antipsychotics at transitions of care are warranted., (© American Society of Health-System Pharmacists 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: In response to the devastating effects of the coronavirus disease 2019 pandemic, several vaccine prototypes have been developed, with the Pfizer/BioNTech (BNT162b2) platform being the first to receive emergency use authorization. Although taken to market on an unprecedented timeline, the safety profile of the drug during clinical trials was shown to be favorable. Shortly after release, reports from the Centers for Disease Control and Prevention demonstrated a higher-than-average rate of anaphylaxis to the vaccine that has been the cause for concern for safety officials and the general public alike. Here, we present a unique case of protracted anaphylaxis in a recipient of the BNT162b2., Case Summary: The patient is a 55-year-old female with a history of multiple allergic reactions who presented with respiratory distress and hives after receiving the first dose of the BNT162b2, despite premedication with IV steroids and diphenhydramine. The refractory nature of her reaction was demonstrated by edema of her tongue (visualized on nasolaryngoscopy), requiring an epinephrine infusion for nearly 3 days. She was discharged from the hospital with instructions not to receive the second dose of the vaccine., Conclusion: Although the exact etiology of anaphylaxis secondary to this messenger RNA-based vaccine is not completely clear, our literature search and review of the patient's course support either polyethylene glycol versus other excipient-related allergy as a possible cause. Based on the protracted nature to our patient's anaphylaxis, critical care management for patients with a true anaphylactic reaction to BNT162b2 may require monitoring for an extended period of time., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

What Is Known and Objective: Sevelamer is an insoluble polymer indicated for the management of hyperphosphatemia in patients with chronic kidney disease (CKD). The package inserts for both tablet formulations recommend the tablets be administered whole. Due to whole tablets being sometimes inadvertently crushed and the significantly increased cost of sevelamer packets, we evaluated the safety and feasibility of crushed sevelamer tablets for enteral feeding tube administration., Methods: A single-centre retrospective chart review was performed. All adult ICU patients prescribed sevelamer carbonate between 1 January 2015 and 31 July 2019 were included if they received at least one dose of a sevelamer tablet or packet, whereas they had an enteral feeding tube in place. The primary outcome was the incidence of an obstructed enteral feeding tube or need for replacement, as defined as the number of occurrences over the total numbers of doses administered. The secondary outcome was the change in phosphorus levels from time of sevelamer initiation to discontinuation or patient discharge., Results: A total of 14 obstructions were reported, four in the tablet arm and ten in the packet arm (0.4% tablet arm, 0.5% packet arm; P = .5931). Of these, four (29%) required tube replacement and were followed by sevelamer discontinuation. Two (14%) were documented to be due to increased tube feeds and esomeprazole. Six (43%) cases required tube replacement, but no issues arose upon continuation. Only one of the obstructions resulted in a recurrent tube occlusion., What Is New and Conclusion: Sevelamer tablets may be crushed and administered via enteral feeding tubes, provided clear instruction on tablet preparation is included. Oral administration in dysphagic patients requires further evaluation with clear protocols for preparation and administration., (© 2020 John Wiley & Sons Ltd.)

Background: Atrial fibrillation (AF) frequently develops during critical illness. In septic shock complicated by rapid AF, the use of phenylephrine may be advantageous secondary to its β-1 sparing properties. However, evidence supporting this strategy is lacking., Objective: The purpose of this study is to determine the clinical effect on rate control of transitioning norepinephrine to phenylephrine in septic shock patients who develop AF with a rapid ventricular response (RVR)., Methods: A single-center retrospective study of septic shock patients admitted to the medical or surgical intensive care unit (ICU) who developed AF with RVR (heart rate >110 beats per minute [bpm]). Patients who were switched to phenylephrine were compared to those who remained on norepinephrine. The primary end point was sustained achievement of rate control. A time-varying Cox proportional hazards model was used to assess the primary end point., Results: A total of 67 patients were included in the study, of which 28 were switched to phenylephrine. Baseline characteristics were similar between groups. The unadjusted hazard ratio for achieving rate control was significant at 1.99 (95% confidence interval [CI]: 1.19-3.34; P < .01) for the phenylephrine group. The adjusted hazard ratio was 1.75 (95% CI: 0.86-3.53; P = .12). There were no statistically significant differences in mortality or ICU length of stay., Conclusion: Our study suggests a potential clinical effect on achieving rate control when switching to phenylephrine cannot be excluded. It remains unclear if there is a benefit on mortality or length of stay outcomes in critically ill patients.

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.

A human colon adenocarcinoma cell line (GC3TK-) was selected for thymidine kinase (TK) deficiency from cloned parental cells (GC3C1) by exposure to 5-bromodeoxyuridine (BrdUrd). The cellular pharmacology of 5-fluorouracil (FUra) and the influence of physiological concentrations of thymidine (dThd; 0.1 to 1 microM) on FUra cytotoxicity during brief exposure in both cell lines were examined. The uptake of FUra during a 1-hr drug exposure, its metabolism to ribo- and deoxyribonucleotides, incorporation into RNA, and inhibition of thymidylate synthase were similar in GC3C1 and GC3TK- cells as were the IC50 values for FUra (26 and 23 microM respectively). TK deficiency did not reduce the intracellular concentrations of FdUMP generated from FUra. In GC3C1, at FUra concentrations up to 100 microM, cytotoxicity was prevented by co-administration of dThd (0.1 to 20 microM). The relationship between cell survival and thymidylate synthase inhibition was close under these conditions. At higher drug concentrations, less dThd protection was observed, and none was detected in GC3TK- cells. Thus, the metabolism of FUra did not appear to be altered substantially in GC3C1 cells selected for TK deficiency. Also in these cells, at concentrations of FUra less than 100 microM, FUra cytotoxicity appeared to be mediated via the inhibition of thymidylate synthase.

In preclinical investigations performed in mice, 2-amino-9-beta-D-ribofuranosyl purine-6-sulfinamide (sulfinosine), a novel derivative of 6-thioguanosine (6TGR), was active against six solid tumors and four strains of experimental leukemia. Sulfinosine penetrated the central nervous system more readily than did 6TGR and, when given repeatedly, was much more effective in the treatment of L1210 leukemia, being curative for some mice. Other findings of major interest to us were the different dosing characteristics of sulfinosine and 6TGR, the divergent efficiencies of the two drugs in generating cellular resistance, and the activity of sulfinosine against experimental leukemias refractory to 6TGR and other experimental or clinically used chemotherapeutic agents. The chemotherapeutic characterization of sulfinosine that evolved from these studies suggests that this agent may have unique properties that deserve clinical consideration. Both the dosing characteristics of the drug and its pronounced activity against thiopurine-resistant experimental leukemia favor the possibility that sulfinosine could be used to advantage in the treatment of human leukemia unresponsive to 6-mercaptopurine or 6-thioguanine.

1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors.

The method for measuring polyglutamate forms of CH2-H4PteGlu and H4PteGlu, by entrapment in ternary complexes with [6-3H]5-fluoro-2'-deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of CH2-H4PteGlu and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relationship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (d) noncovalent complexes formed with PteGlu2-5 could be isolated only at concentrations considerably higher than those required for CH2-H4PteGlu1-6, and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of CH2-H4PteGlu and the combined pools of CH2-H4PteGlu plus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of CH2-H4PteGlu and H4PteGlu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the CH2-H4PteGlu pool, and greater than 93% of the combined pools. A small percentage of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of CH2-H4PteGlu and H4PteGlu was estimated in the range of 0.5 to 2.7 microM in cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 microM. Using thymidylate synthase purified from colon adenocarcinoma HxVRC5, CH2-H4PteGlu5 (where the subscript digit attached to the glutamate portion equals the number of glutamate residues) stabilized the covalent ternary complex at greater than 200-fold lower concentration in comparison to CH2-H4PteGlu1. Data indicated that in each colon tumor, the concentrations of CH2-H4PteGlun or CH2-H4PteGlun plus H4PteGlun were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5-fluorouracil. These data support therapeutic modulation to increase the concentration of CH2-H4PteGlun in the treatment of colon adenocarcinomas with 5-fluorouracil.

Steroid hormone receptors, including those for vitamin D3, contain reactive sulfhydryl group(s) essential for hormone binding. On this basis, several compounds that are capable of interacting with sulfhydryl groups were tested for their ability to dissociate 1,25-dihydroxyvitamin D3 from chicken intestinal receptors. At concentrations resulting in 50% displacement of specifically bound cytoplasmic 1,25-dihydroxyvitamin D3, the order of potency for these displacing reagents is mersalyl acid greater than sodium thiocyanate approximately equal to p-hydroxymercuribenzoate approximately equal to mercuric chloride much greater than 5,5'-dithiobis-(2-nitrobenzoic acid). Hormone displacement by mersalyl acid and p-hydroxymercuribenzoate was completely reversible upon dithiothreitol addition. In contrast limited rebinding of 1,25-dihydroxyvitamin D3 occurred after, 5,5-dithiobis-(2-nitrobenzoic acid) and mercuric chloride treatment. Furthermore, at least for mersalyl acid treatment, hormone displacement and subsequent regeneration of sterol binding did not seem to alter the integrity of the receptor as evidenced by sucrose gradient analysis, DNA-cellulose and phosphocellulose chromatography. Additionally, treatment of the nuclear 1,25-dihydroxyvitamin D3 receptor did not significantly affect the apparent equilibrium dissociation constant for hormone binding (Kd = 2.7 X 10(-10) M; Kd = 1.7 X 10(-10) M, for control and mersalyl acid treated receptor, respectively). Finally, a method has been developed for measurement of occupied 1,25-dihydroxyvitamin D3 receptors. The unfilled receptors were quantitated in the cytoplasm or chromatin extracted fraction by incubation with radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 3-6 h without interference from previously filled sites. The total receptor measurement is carried out by incubation of cytosol or nuclear extract with 0.5-1.0 mM mersalyl acid for 1.0 h. Exchange of radioactive sterol for the bound nonradioactive sterol is accomplished by incubation with 1-2 mM dithiothreitol and 1.0 nM radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 16 h. Subtracting the unfilled sites from total sites results in a measurement of filled sites. With this exchange assay, estimates of total receptor in untreated and 1,25-dihydroxyvitamin D3 treated chicks (estimated 2 h after injection of doses 0.3-300 nmol) were not significantly different (66.2 +/- 4.5 versus 64.4 +/- 10.4 to 69.0 +/- 5.9 pg/mg protein, respectively). Furthermore, quantitation of total receptor by direct or exchange assay obtained from in vitro incubations of intestinal slices with radioactive and nonradioactive 1,25-dihydroxyvitamin D3 were not

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of less than 3 microM. The interaction of polyglutamates of [6R]CH2-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 microM) were greater than 200-fold and [6R]CH2-H4PteGlu2 (18.2 microM) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 microM) at stabilizing ternary complexes for a T1/2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 microM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 x 10(-8) M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 x 10(-9) M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 x 10(6) M-1 min-1 for 10 or 20 microM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 microM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n greater than or equal to 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Growth of human adenocarcinomas of the colon and rectum in immunoincompetent mice has allowed for a greater understanding of the interaction of 5-fluorouracil, its metabolism, and mechanism(s) of cytotoxicity under conditions of tumor growth in situ. Conversely, this agent has proven to be a useful tool in defining metabolic characteristics in human colon adenocarcinomas. Analysis of tumor sensitivity to 5-fluorouracil (FUra),5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd) suggests that growth inhibition in vivo is related to a DNA-directed event. Resistance, de novo appears to be a consequence of relatively transient inhibition of the target enzyme thymidylate synthase (dTMP-synthase), which may be a consequence of low concentrations of 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) or its polyglutamate forms within tumor cells in situ. In order to study the relationship between inhibition of dTMP-synthase and growth inhibition, mutant cells deficient in their ability to salvage dThd have been selected, and grown as xenografts. Data suggest that transient inhibition of dTMP-synthase and not dThd salvage is responsible for resistance de novo, and that prolonged inhibition of dTMP-synthase would be a lethal event in vivo. This would predict that a cell lacking dTMP-synthase activity would not be tumorigenic. This has been tested directly by selecting clones of GC3 colon adenocarcinoma cells deficient in dTMP-synthase (TS-) activity. Preliminary data indicate that each of 3 TS- clones is tumorigenic in athymic nude mice. The importance of dTMP-synthase as a target for drug development is discussed with respect to these findings.

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.

The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetrahydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) absorbed greater than 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37 degrees), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.