Monoclonal Antibodies Engineered with Fc Region Mutations to Extend Protection against Fentanyl Toxicity.

Fentanyl and other synthetic opioids are the leading cause of drug-related deaths in the United States. mAbs that selectively target fentanyl and fentanyl analogues offer a promising strategy for treating both opioid-related overdoses and opioid use disorders. To increase the duration of efficacy of a candidate mAb against fentanyl, we selected three sets of mutations in the Fc region of an IgG1 anti-fentanyl mAb (HY6-F9DF215, HY6-F9DHS, HY6-F9YTE) to increase binding to the neonatal Fc receptor (FcRn). The mAb mutants were compared against unmodified (wild-type [WT], HY6-F9WT) anti-fentanyl mAb for fentanyl binding, thermal stability, and FcRn affinity in vitro, and for efficacy against fentanyl and mAb half-life in vivo in mice. Biolayer interferometry showed a >10-fold increase in the affinity for recombinant FcRn of the three mutant mAbs compared with HY6-F9WT. During an acute fentanyl challenge in mice, all FcRn-mutated mAbs provided equal protection against fentanyl-induced effects, and all mAbs reduced brain fentanyl levels compared with the saline group. Serum persistence of the mutant mAbs was tested in Tg276 transgenic mice expressing human FcRn. After administration of 40 mg/kg HY6-F9WT, HY6-F9DF215, HY6-F9DHS, and HY6-F9YTE, the mAbs showed half-lives of 6.3, 26.4, 14.7, and 6.9 d, respectively. These data suggest that modification of mAbs against fentanyl to bind to FcRn with higher affinity can increase their half-life relative to WT mAbs while maintaining efficacy against the toxic effects of fentanyl, further supporting their potential role as a therapeutic treatment option for opioid use disorder and overdose.

Evaluating the rate of reversal of fentanyl-induced respiratory depression using a novel long-acting naloxone nanoparticle, cNLX-NP.

Introduction: Fentanyl and fentanyl analogs (F/FA) have become increasingly common adulterants in counterfeit prescription pills and illicit street drug mixtures due to their ease of synthesis and exceedingly high potency. The ongoing epidemic of fatal overdoses fueled by F/FA continues to highlight the need for longer-acting therapies than naloxone (NLX), the current gold-standard for reversing opioid overdoses, which shows limited efficacy to prevent renarcotization associated with F/FA toxicity. A novel opioid reversal agent based on covalent naloxone nanoparticles (cNLX-NP) has been shown to blunt fentanyl-induced respiratory depression out to 48 hr, demonstrating its potential therapeutic utility. The purpose of this study was to characterize how rapidly cNLX-NP reverses fentanyl-induced respiratory effects as well as the duration of its protective effects. Methods: Sprague Dawley male rats (n=6/group) were tested on an oximeter for baseline percent arterial oxygen saturation (%SaO2) challenged with 0.1 mg/kg SC fentanyl and 15 min later given 10 mg/kg IM doses of NLX, nalmefene (NLMF), or cNLX-NP and continuously monitored via oximetry for 10 minutes. One week later the experiment was repeated using a 1:1 mixture of NLX:cNLX-NP as the reversal agent in the rats that previously received NLX alone. Results: While both NLX and NLMF rapidly reversed %SaO2 to baseline within 1 min, rats that received cNLX-NP did not return to >90% SaO2 levels until 9 min after administration. Similarly, heart and breath rates returned to baseline within 1 min of treatment with NLX and NLMF but did not return to baseline until 10 minutes after cNLX-NP administration. In contrast, NLX:cNLX-NP reversed all fentanyl-induced respiratory depressive effects within one minute. Discussion: While cNLX-NP alone may not sufficiently reverse F/FA overdose in a timely manner, mixing free NLX with cNLX-NP can provide a mechanism to both rapidly reverse fentanyl-related effects and maintain extended protection against synthetic opioid toxicity. These data support further development of cNLX-NP as a fast-acting and long-lasting antidote to treat F/FA-induced respiratory depression and overdose, and potentially prevent renarcotization in humans.

Multivalent Vaccination Strategies Protect against Exposure to Polydrug Opioid and Stimulant Mixtures in Mice and Rats.

Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among occasional users, and those with either opioid use disorder (OUD) or substance use disorder (SUD). As a complementary strategy to current pharmacotherapies, active immunization with conjugate vaccines has been proposed as a viable intervention to treat OUD as well as other SUD for which there are either limited or no treatment options. Vaccination against opioids and stimulants could help address the limitations of current medications (e.g., patient access, compliance, misuse liability, and safety) by providing an additional tool to prevent drug misuse and/or overdoses. However, more research is needed to fully understand the potential benefits and limitations of using vaccines to treat SUD and overdose and to inform us on how to deploy this strategy in the field. Previous reports have shown promise by combining two vaccines into bivalent vaccine formulations to concurrently target multiple drugs. Here, multiple individual candidate monovalent vaccines were incrementally combined in multivalent vaccine formulations to simultaneously target fentanyl, carfentanil, oxycodone, heroin, methamphetamine, and their analogs or metabolites. Bi-, tri-, and quadrivalent vaccine formulations induced the formation of independent serum antibody responses against their respective opioid targets and selectively attenuated the distribution of each individual drug to the brain in mice and rats. Results indicate that a single injection of an admixed multivalent vaccine formulation may be more effective than coinjecting multiple monovalent vaccines at multiple sites. Finally, adding a methamphetamine conjugate vaccine to an quadrivalent opioid vaccine in a pentavalent formulation did not interfere with the production of effective antiopioid IgG antibodies. Multivalent vaccines could provide multifaceted, yet selective, protection against polydrug use and exposure.

Anti-Strychnine Immunoconjugate Reduces the Effects of Strychnine-Induced Toxicity in Mice.

Strychnine poisoning induces seizures that result in loss of control of airway muscles, leading to asphyxiation and subsequent death. Current treatment options are limited, requiring hands-on medical care and isolation to low-stimulus environments. Anticonvulsants and muscle relaxants have shown limited success in cases of severe toxicity. Furthermore, nonfatal strychnine poisoning is likely to result in long-term muscular and cognitive damage. Due to its potency, accessibility, and lack of effective antidotes, strychnine poses a unique threat for mass casualty incidents. As a first step toward developing an anti-strychnine immunotherapy to reduce or prevent strychnine-induced seizures, a strychnine vaccine was synthesized using subunit keyhole limpet hemocyanin. Mice were vaccinated with the strychnine immunoconjugate and then given a 0.75 mg/kg IP challenge of strychnine and observed for seizures for 30 min. Vaccination reduced strychnine-induced events, and serum strychnine levels were increased while brain strychnine levels were decreased in vaccinated animals compared to the control. These data demonstrate that strychnine-specific antibodies can block the seizure-inducing effects of strychnine and could be used to develop a therapeutic for strychnine poisoning.

Pre-clinical safety and toxicology profile of a candidate vaccine to treat oxycodone use disorder.

Opioid use disorders (OUD) and overdose represent a public health threat, resulting in thousands of deaths annually worldwide. Vaccines offer a promising treatment for OUD and potentially the prevention of fatal overdoses. The Oxy(Gly)4-sKLH Conjugate Vaccine, Adsorbed (Oxy(Gly)4-sKLH) has shown promising pre-clinical efficacy at reducing the behavioral and pharmacological effects of oxycodone. To support its clinical evaluation, a GLP toxicology study was performed to address the safety of Oxy(Gly)4-sKLH. Sprague Dawley rats were vaccinated with either aluminum adjuvant (alum) or vaccine adsorbed on alum. Low and high doses of Oxy(Gly)4-sKLH, equivalent to a 1X or 47X human dose, respectively, were administered every two weeks for a total of four vaccinations. Both vaccine doses induced high antibody titers. Vaccine-related toxicity was assessed postmortem in one experimental group after receiving the fourth immunization of the vaccine's high dose. For the remaining experimental groups, rats were challenged with 1.5 mg/kg/day s.c. oxycodone for 7 days after the fourth vaccination to assess whether concurrent exposure to oxycodone in vaccinated animals resulted in toxicity. All rats, except a subset of the aluminum control and the high dose vaccine groups, were sacrificed following oxycodone exposure. These subsets were allowed a four weeks recovery period prior to euthanasia. In this study, no Oxy(Gly)4-sKLH-related hematology, clinical chemistry, urinalysis, body weight, organ weight, or anatomic pathology toxicological findings were observed. These results demonstrate that the Oxy(Gly)4-sKLH vaccine is well tolerated, is immunogenic even at low doses, and does not produce undesired side effects in rats.

Covalently Loaded Naloxone Nanoparticles as a Long-Acting Medical Countermeasure to Opioid Poisoning.

The mu opioid receptor antagonist naloxone has been a vital, long-standing countermeasure in the ongoing battle against opioid use disorders (OUD) and toxicity. However, due to its distinctive short elimination half-life, naloxone has shown diminished efficacy in cases of synthetic opioid poisoning as larger or repeated doses of the antidote have been required to achieve adequate reversal of severe respiratory depression and prevent episodes of renarcotization. This report describes the synthesis, characterization, and in vivo evaluation of a novel, nanoparticle-based naloxone formulation that provides extended protection against the toxic effects of the powerful synthetic opioid fentanyl. The strategy was predicated on a modified two-step protocol involving the synthesis and subsequent nanoprecipitation of a poly(lactic-co-glycolic acid) polymer scaffold bearing a covalently linked naloxone chain end (drug loading ∼7% w/w). Pharmacokinetic evaluation of the resulting covalently loaded naloxone nanoparticles (cNLX-NP) revealed an elimination half-life that was 34 times longer than high dose free naloxone (10 mg/kg) in male Sprague-Dawley rats. This enhancement was further demonstrated by cNLX-NP in subsequent in vivo studies affording protection against fentanyl-induced respiratory depression and antinociception for up to 48 h following a single intramuscular injection. These discoveries support further investigation of cNLX-NP as a potential therapeutic to reverse overdose and prevent renarcotization from fentanyl and its potent analogs.

Attenuating nicotine's effects with high affinity human anti-nicotine monoclonal antibodies.

Use of nicotine-specific monoclonal antibodies (mAbs) to sequester and reduce nicotine distribution to brain has been proposed as a therapeutic approach to treat nicotine addiction (the basis of tobacco use disorder). A series of monoclonal antibodies with high affinity for nicotine (nic•mAbs) was isolated from B-cells of vaccinated smokers. Genes encoding 32 unique nicotine binding antibodies were cloned, and the mAbs expressed and tested by surface plasmon resonance to determine their affinity for S-(-)-nicotine. The highest affinity nic•mAbs had binding affinity constants (KD) between 5 and 67 nM. The 4 highest affinity nic•mAbs were selected to undergo additional secondary screening for antigen-specificity, protein properties (including aggregation and stability), and functional in vivo studies to evaluate their capacity for reducing nicotine distribution to brain in rats. The 2 most potent nic•mAbs in single-dose nicotine pharmacokinetic experiments were further tested in a dose-response in vivo study. The most potent lead, ATI-1013, was selected as the lead candidate based on the results of these studies. Pretreatment with 40 and 80 mg/kg ATI-1013 reduced brain nicotine levels by 56 and 95%, respectively, in a repeated nicotine dosing experiment simulating very heavy smoking. Nicotine self-administration was also significantly reduced in rats treated with ATI-1013. A pilot rat 30-day repeat-dose toxicology study (4x200mg/kg ATI-1013) in the presence of nicotine indicated no drug-related safety concerns. These data provide evidence that ATI-1013 could be a potential therapy for the treatment of nicotine addiction.

Combining a Candidate Vaccine for Opioid Use Disorders with Extended-Release Naltrexone Increases Protection against Oxycodone-Induced Behavioral Effects and Toxicity.

Opioid use disorders (OUDs) and opioid-related fatal overdoses are a significant public health concern in the United States and worldwide. To offer more effective medical interventions to treat or prevent OUD, antiopioid vaccines are in development that reduce the distribution of the targeted opioids to brain and subsequently reduce the associated behavioral and toxic effects. It is of critical importance that antiopioid vaccines do not interfere with medications that treat OUD. Hence, this study tested the preclinical proof of concept of combining a candidate oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with an FDA-approved extended-release naltrexone (XR-NTX) depot formulation in rats. The effects of XR-NTX on oxycodone-induced motor activity and antinociception were first assessed in nonvaccinated naïve rats to establish a baseline for subsequent studies. Next, OXY-KLH and XR-NTX were coadministered to determine whether the combination would affect the efficacy of each individual treatment, and it was found that the combination of OXY-KLH and XR-NTX offered greater efficacy in reducing oxycodone-induced motor activity, thigmotaxis, antinociception, and respiratory depression over a range of repeated or escalating oxycodone doses in rats. These data support the feasibility of combining antibody-based therapies with opioid receptor antagonists to provide greater or prolonged protection against opioid-related toxicity or overdose. Combining antiopioid vaccines with XR-NTX may provide prophylactic measures to subjects at risk of relapse and accidental or deliberate exposure. Combination therapy may extend to other biologics (e.g., monoclonal antibodies) and medications against substance use disorders. SIGNIFICANCE STATEMENT: Opioid use disorders (OUDs) remain a major problem worldwide, and new therapies are needed. This study reports on the combination of an oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with a currently approved OUD therapy, extended-release naltrexone (XR-NTX). Results demonstrated that XR-NTX did not interfere with OXY-KLH efficacy, and combination of low doses of XR-NTX with vaccine was more effective than each individual treatment alone to reduce behavioral and toxic effects of oxycodone, suggesting that combining OXY-KLH with XR-NTX may improve OUD outcomes.

A Fentanyl Vaccine Alters Fentanyl Distribution and Protects against Fentanyl-Induced Effects in Mice and Rats.

Fentanyl is an extremely potent synthetic opioid that has been increasingly used to adulterate heroin, cocaine, and counterfeit prescription pills, leading to an increase in opioid-induced fatal overdoses in the United States, Canada, and Europe. A vaccine targeting fentanyl could offer protection against the toxic effects of fentanyl in both recreational drug users and others in professions at risk of accidental exposure. This study focuses on the development of a vaccine consisting of a fentanyl-based hapten (F) conjugated to keyhole limpet hemocyanin (KLH) carrier protein or to GMP-grade subunit KLH (sKLH). Immunization with F-KLH in mice and rats reduced fentanyl-induced hotplate antinociception, and in rats reduced fentanyl distribution to the brain compared with controls. F-KLH did not reduce the antinociceptive effects of equianalgesic doses of heroin or oxycodone in rats. To assess the vaccine effect on fentanyl toxicity, rats immunized with F-sKLH or unconjugated sKLH were exposed to increasing subcutaneous doses of fentanyl. Vaccination with F-sKLH shifted the dose-response curves to the right for both fentanyl-induced antinociception and respiratory depression. Naloxone reversed fentanyl effects in both groups, showing that its ability to reverse respiratory depression was preserved. These data demonstrate preclinical selectivity and efficacy of a fentanyl vaccine and suggest that vaccines may offer a therapeutic option in reducing fentanyl-induced side effects.

The nicotine-degrading enzyme NicA2 reduces nicotine levels in blood, nicotine distribution to brain, and nicotine discrimination and reinforcement in rats.

BACKGROUND: The bacterial nicotine-degrading enzyme NicA2 isolated from P. putida was studied to assess its potential use in the treatment of tobacco dependence. RESULTS: Rats were pretreated with varying i.v. doses of NicA2, followed by i.v. administration of nicotine at 0.03 mg/kg. NicA2 had a rapid onset of action reducing blood and brain nicotine concentrations in a dose-related manner, with a rapid onset of action. A 5 mg/kg NicA2 dose reduced the nicotine concentration in blood by > 90% at 1 min after the nicotine dose, compared to controls. Brain nicotine concentrations were reduced by 55% at 1 min and 92% at 5 min post nicotine dose. To evaluate enzyme effects at a nicotine dosing rate equivalent to heavy smoking, rats pretreated with NicA2 at 10 mg/kg were administered 5 doses of nicotine 0.03 mg/kg i.v. over 40 min. Nicotine levels in blood were below the assay detection limit 3 min after either the first or fifth nicotine dose, and nicotine levels in brain were reduced by 82 and 84%, respectively, compared to controls. A 20 mg/kg NicA2 dose attenuated nicotine discrimination and produced extinction of nicotine self-administration (NSA) in most rats, or a compensatory increase in other rats, when administered prior to each daily NSA session. In rats showing compensation, increasing the NicA2 dose to 70 mg/kg resulted in extinction of NSA. An enzyme construct with a longer duration of action, via fusion with an albumin-binding domain, similarly reduced NSA in a 23 h nicotine access model at a dose of 70 mg/kg. CONCLUSIONS: These data extend knowledge of NicA2's effects on nicotine distribution to brain and its ability to attenuate addiction-relevant behaviors in rats and support its further investigation as a treatment for tobacco use disorder.

Opioid Dose- and Route-Dependent Efficacy of Oxycodone and Heroin Vaccines in Rats.

Heroin and oxycodone abuse occurs over a wide range of drug doses and by various routes of administration characterized by differing rates of drug absorption. The current study addressed the efficacy of a heroin vaccine [morphine hapten conjugated to keyhole limpet hemocyanin (M-KLH)] or oxycodone vaccine [oxycodone hapten conjugated to keyhole limpet hemocyanin (OXY-KLH)] for reducing drug distribution to brain after intravenous heroin or oxycodone, or subcutaneous oxycodone. Rats immunized with M-KLH or keyhole limpet hemocyanin (KLH) control received an intravenous bolus dose of 0.26 or 2.6 mg/kg heroin. Vaccination with M-KLH increased retention of heroin and its active metabolites 6-acetylmorphine (6-AM) and morphine in plasma compared with KLH controls, and reduced total opioid (heroin + 6-AM + morphine) distribution to brain but only at the lower heroin dose. Immunization also protected against respiratory depression at the lower heroin dose. Rats immunized with OXY-KLH or KLH control received 0.22 or 2.2 mg/kg oxycodone intravenously, the molar equivalent of the heroin doses. Immunization with OXY-KLH significantly reduced oxycodone distribution to brain after either oxycodone dose, although the magnitude of effect of immunization at the higher oxycodone dose was small (12%). By contrast, vaccination with OXY-KLH was more effective when oxycodone was administered subcutaneously rather than intravenously, reducing oxycodone distribution to brain by 44% after an oxycodone dose of 2.3 mg/kg. Vaccination also reduced oxycodone-induced antinociception. These data suggest that the efficacy of OXY-KLH and M-KLH opioid vaccines is highly dependent upon opioid dose and route of administration.

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

Engineering of a hybrid nanoparticle-based nicotine nanovaccine as a next-generation immunotherapeutic strategy against nicotine addiction: A focus on hapten density.

Although vaccination is a promising way to combat nicotine addiction, most traditional hapten-protein conjugate nicotine vaccines only show limited efficacy due to their poor recognition and uptake by immune cells. This study aimed to develop a hybrid nanoparticle-based nicotine vaccine with improved efficacy. The focus was to study the impact of hapten density on the immunological efficacy of the proposed hybrid nanovaccine. It was shown that the nanovaccine nanoparticles were taken up by the dendritic cells more efficiently than the conjugate vaccine, regardless of the hapten density on the nanoparticles. At a similar hapten density, the nanovaccine induced a significantly stronger immune response against nicotine than the conjugate vaccine in mice. Moreover, the high- and medium-density nanovaccines resulted in significantly higher anti-nicotine antibody titers than their low-density counterpart. Specifically, the high-density nanovaccine exhibited better immunogenic efficacy, resulting in higher anti-nicotine antibody titers and lower anti-carrier protein antibody titers than the medium- and low-density versions. The high-density nanovaccine also had the best ability to retain nicotine in serum and to block nicotine from entering the brain. These results suggest that the hybrid nanoparticle-based nicotine vaccine can elicit strong immunogenicity by modulating the hapten density, thereby providing a promising next-generation immunotherapeutic strategy against nicotine addiction.

A nanoparticle-based nicotine vaccine and the influence of particle size on its immunogenicity and efficacy.

Traditional hapten-protein conjugate nicotine vaccines have shown less than desired immunological efficacy due to their poor recognition and internalization by immune cells. We developed a novel lipid-polymeric hybrid nanoparticle-based nicotine vaccine to enhance the immunogenicity of the conjugate vaccine, and studied the influence of particle size on its immunogenicity and pharmacokinetic efficacy. The results demonstrated that the nanovaccines, regardless of size, could induce a significantly stronger immune response against nicotine compared to the conjugate vaccine. Particularly, a significantly higher anti-nicotine antibody titer was achieved by the 100 compared to the 500nm nanovaccine. In addition, both the 100 and 500nm nanovaccines reduced the distribution of nicotine into the brain significantly. The 100nm nanovaccine exhibited better pharmacokinetic efficacy than the 500nm nanovaccine in the presence of alum adjuvant. These results suggest that a lipid-polymeric nanoparticle-based nicotine vaccine is a promising candidate to treat nicotine dependence.

Pharmacokinetic correlates of the effects of a heroin vaccine on heroin self-administration in rats.

The purpose of this study was to evaluate the effects of a morphine-conjugate vaccine (M-KLH) on the acquisition, maintenance, and reinstatement of heroin self-administration (HSA) in rats, and on heroin and metabolite distribution during heroin administration that approximated the self-administered dosing rate. Vaccination with M-KLH blocked heroin-primed reinstatement of heroin responding. Vaccination also decreased HSA at low heroin unit doses but produced a compensatory increase in heroin self-administration at high unit doses. Vaccination shifted the heroin dose-response curve to the right, indicating reduced heroin potency, and behavioral economic demand curve analysis further confirmed this effect. In a separate experiment heroin was administered at rates simulating heroin exposure during HSA. Heroin and its active metabolites, 6-acetylmorphine (6-AM) and morphine, were retained in plasma and metabolite concentrations were reduced in brain in vaccinated rats compared to controls. Reductions in 6-AM concentrations in brain after vaccination were consistent with the changes in HSA rates accompanying vaccination. These data provide evidence that 6-AM is the principal mediator of heroin reinforcement, and the principal target of the M-KLH vaccine, in this model. While heroin vaccines may have potential as therapies for heroin addiction, high antibody to drug ratios appear to be important for obtaining maximal efficacy.

Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats.

Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105% of the target value. Variability was 4.0-8.9% for all analytes within the range of 5-200 ng/mL for heroin, 5-1000 ng/mL for 6-MAM, and 10-200 ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4 mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80°C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3% over one week in rat plasma under these conditions, and by <1-4.7% in bovine serum or human plasma.

Twinning in a colony of vervet monkeys (Cercopithecus aethiops sabaeus).

From 1975 through 1991, three sets of twins were born from a total of 693 live and stillborn births (0.43%) at the UCLA/Sepulveda Veterans Administration Medical Center Nonhuman Primate Research Laboratory, CA. None of the twin's mothers were related. Positive patrilineal relationships have not been established; however, a brother (not a twin) of the first set of twins may have fathered the third set. All twins were born to multiparous females and, in each case, only one infant survived beyond 4 days. One set of twins was dizygotic; the genetic status of the others is unknown. © 1994 Wiley-Liss, Inc.

Personality features in vervet monkeys: The effects of sex, age, social status, and group composition.

Personality ratings were completed in 97 socially living vervet monkeys (Cercopithecus aethiops sabaeus) ranging in age from 19 months to 14 years. Monkeys were rated on 17 distinct items on three separate occasions over a 14 month period. For each rating session, factor analysis resulted in similar factors: socially competent, playful/curious, and opportunistic, which are provisionally labeled as personality features. Individuals' scores on the three factors were linked to the subjects' social status, age, and sex. Relative to subordinate animals, dominant individuals had higher scores on the socially competent and lower scores on the opportunistic factors. Juvenile and subadult animals scored higher on playful/curious and opportunistic factors than adults. Females had higher scores on the opportunistic factor than males. Scores on the three factors changed during maturation, which suggests that some aspects of personality are age- and context-dependent. When group composition and social status were unaltered, intraindividual differences in factor scores were as consistent over time as were the rates of social grooming, being in proximity to others, resting, and other overt behaviors. Alterations in social status, group composition, and group membership were associated with changes in factor scores among some, but not all animals. These observations suggest that studies assessing personality can supplement ethological investigations of nonhuman primate behavior, and support the view that nonhuman Primates can serve as models for important features of human behavior. © 1994 Wiley-Liss, Inc.

Rank, relationships, and responses to intruders among adult male vervet monkeys.

We examined the influences of dyadic relationships among captive adult male vervet monkeys (Cercopithecus aethiops sabaeus) on behavior directed toward caged "intruder" males placed inside subjects' enclosures. Subjects were all 9 adult male residents from three stable social groups, each of which contained 3 adult males, at least 3 adult females, and their immature offspring. Every male was observed in two 3-hour sessions, each time with one of the 2 other adult males from his group. Observation sessions consisted of six consecutive 30-min stages in which group composition and the presence of the intruder were manipulated. All groups exhibited a stable, linear male dominance hierarchy prior to and throughout the study. In each group, there was one pair of males, when together, in which each member exhibited higher rates of intruder-directed approach and aggressive behaviors than when either animal was paired with the third male of his social group. Such pairs were also distinguished by high levels of within-pair agonistic interactions. The higher-ranking member of each dyad was the most aggressive male toward the intruder in his social group, although only one of these animals was the dominant male of his group. Mutual facilitation of aggression against intruding males is interpreted as cooperative behavior benefitting both males by increasing the likelihood of repelling a potential competitor for resident females. Such cooperation provides further evidence in nonhuman primates for cohesive male-male dyads between animals whose social interactions are characterized by agonism. © 1993 Wiley-Liss, Inc.