Daniel E. Keyler

Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and effects on nicotine distribution to brain

Vaccination against nicotine has been proposed as a potential treatment for nicotine dependence. Because vaccination may take months to elicit satisfactory antibody levels, the clinical usefulness of this approach will be enhanced if vaccination can be accomplished during continued nicotine intake (e.g., before a smoker quits). The current study examined the immunogenicity of a nicotine conjugate vaccine during continued nicotine dosing in rats, and its effects on nicotine distribution to brain. In the first experiment, nicotine was administered over 11 weeks as 20 intra venous (i.v.) bolus injections per day during the rats active cycle to simulate the usual pattern of nicotine intake from cigarette smoking. In the second experiment, rats received a continuous s.c. infusion of nicotine by osmotic pump for 11 weeks to provide serum nicotine concentrations equivalent to those of a heavy smoker and 24 h/day nicotine exposure. Nicotine-specific antibody titers after the third booster dose were not compromised by either regimen of concurrent nicotine administration compared to those of rats receiving saline. A single additional i.v. nicotine dose was administered at the end of each experiment. The distribution of this single nicotine dose to brain was reduced by 40-60% in vaccinated rats compared to controls. Vaccine efficacy in reducing nicotine distribution to brain was not compromised by concurrent nicotine administration. These data suggest that vaccination during concurrent nicotine administration is feasible, and that the ability of vaccination to reduce nicotine distribution to brain is preserved even after months of nicotine dosing at rates approximating cigarette smoking.

Tissue-dependent effects of immunization with a nicotine conjugate vaccine on the distribution of nicotine in rats.

Vaccination of rats against nicotine reduces nicotine distribution to brain even at nicotine doses greatly exceeding the estimated binding capacity of the available antibody. This observation suggests a differential effect by which vaccination reduces nicotine distribution to brain to a greater extent than to other tissues. To test this hypothesis, vaccinated rats received a single intravenous nicotine dose equal to twice the estimated binding capacity of nicotine-specific antibody in vaccinated rats. The total and bound serum nicotine concentrations were higher in the vaccinated rats compared to controls, while the unbound serum nicotine concentration was lower. Distribution of nicotine to brain was reduced in vaccinated rats in a time-dependent manner, with a greater reduction at 1 min (64%) than at 25 min (45%). Vaccination reduced nicotine distribution to muscle, testis, spleen, liver, heart, and kidney, but to a lesser extent than to brain, while nicotine distribution to fat was increased. Chronically infused nicotine showed a similarly altered pattern of tissue distribution in vaccinated rats, but differences were in general smaller than after a single nicotine dose; brain nicotine concentration was 24% lower in vaccinated rats, while lung nicotine concentration was higher. The presence of nicotine-specific antibody in tissues may have contributed to the increased nicotine concentrations in fat and lung. These data suggest that vaccination reduces nicotine distribution to brain not only by sequestering nicotine in serum but also by redirecting tissue distribution disproportionately away from brain, such that nicotine concentrations are reduced to a greater extent in brain than in other tissues.

Inhibition of nicotine-induced seizures in rats by combining vaccination against nicotine with chronic nicotine infusion.

The ability of a nicotine vaccine to protect against nicotine-induced seizures was studied in rats. Groups of 10 rats were vaccinated with 3 doses of either a nicotine conjugate vaccine over 6 weeks to elicit high titers of nicotine-specific antibodies or with a control vaccine. Rats were then pretreated with a 1-week subcutaneous infusion of either nicotine 1 mg/kg/day or saline and then received a single 2 mg/kg ip dose of nicotine to provoke seizures. Vaccination reduced the incidence of seizures. The combination of vaccination and pretreatment with nicotine infusion was more effective than either treatment alone. These data suggest that vaccination is protective against this toxic effect of nicotine and that combining vaccination and chronic nicotine administration may provide a novel strategy for blocking some effects of nicotine.

Reduction of Desipramine Cardiotoxicity and Prolongation of Survival in Rats With the Use of Polyclonal Drug-Specific Antibody Fab Fragments☆☆☆★★★

STUDY OBJECTIVE Tricyclic antidepressants (TCAs) are a leading cause of death from intentional drug overdose. Available therapies are often unsatisfactory. In this study we evaluated the use of a high-affinity drug-specific polyclonal Fab fragment (TFab) as an antidote to desipramine toxicity. DESIGN We gave anaesthetized rats under mechanical ventilation IV desipramine so that we might study the effect of TFab on survival or IP desipramine to facilitate study of the interaction of TFab and hypertonic sodium bicarbonate (NaHCO3), the standard clinical treatment for TCA overdose. INTERVENTIONS For the study of the effects of TFab and NaHCO3 on survival, each rat was given a constant IV infusion of desipramine until it died, together with TFab 2 g/kg, bovine serum albumin, or .9% NaCl starting 5 minutes after the desipramine infusion. In the study of the interaction of TFab and NaHCO3, each rat received 30 mg/kg IP desipramine followed by TFab (molar TFab:desipramine ratio, .11), NaHCO3, TFab+NaHCO3, or NaCl at the time of maximal toxicity (15 minutes). RESULTS In the survival protocol, QRS-interval duration, systolic blood pressure, and heart rate were significantly improved by TFab, and survival was prolonged by 58% compared with that in the albumin and NaCl groups (P < .001). The molar ratio of TFab to administered desipramine was .21. The unbound fraction of desipramine in serum at the time of death was reduced by TFab, but the unbound desipramine concentration was not, suggesting that TFab prolonged survival by delaying the increase in the unbound serum desipramine concentration. In the interaction protocol, neither TFab nor NaHCO3 was effective alone, but the combination significantly reduced QRS-interval prolongation (P = .001). CONCLUSION These data demonstrate the efficacy of TFab in reducing desipramine-induced cardiovascular toxicity and prolonging survival. The pharmacokinetic effects of TFab in rats with severe desipramine toxicity were similar to those observed in sublethal desipramine toxicity. Therapeutic benefit is enhanced by the concurrent use of NaHCO3 and may be achieved despite binding only a fraction of the desipramine dose.

Hypertonic sodium bicarbonate partially reverses QRS prolongation due to flecainide in rats

Hypertonic (1M) sodium bicarbonate can partially reverse the cardiac toxicity of some Class IA antiarrhythmic agents, presumably by antagonizing sodium channel inhibition. We studied the effects of 1M sodium bicarbonate on toxicity due to the Class IC drug flecainide. Anesthetized rats received i.v. loading and maintenance doses of flecainide to produce QRS prolongation of 76% that was stable over the 60 min study period. 20 min after the start of the maintenance infusion, groups of 8 rats received an i.v. infusion of 1M sodium bicarbonate (6 meq/kg) or an equal volume of 0.9% saline. QRS prolongation was reduced by 1M sodium bicarbonate but not only 0.9% saline (% change -12.2 +/- 10.0 v. +3.0 +/- 2.7, p = 0.001). Expressed as a percent of the flecainide-induced QRS prolongation, bicarbonate reduced this prolongation by 26.5%. The improvement in QRS duration persisted until sacrifice 30 min later. Compared to controls, the bicarbonate group had a significantly higher blood pH (7.55 +/- 0.06 v. 7.44 +/- 0.05) and serum sodium concentration (149 +/- 5 v. 137 +/- 2 meq/l). Serum flecainide concentrations were similar. These data suggest that 1M sodium bicarbonate can partially reverse flecainide-induced conduction delay in rats. This effect may be due to changes in the extracellular pH and sodium concentration. 1M sodium bicarbonate may be useful in assessing the role of sodium channel inhibition in mediating the toxicity of flecainide or other Class IC drugs.

Replies to Fry et al. (Toxicon 2012, 60/4, 434-448). Part A. Analyses of squamate reptile oral glands and their products: A call for caution in formal assignment of terminology designating biological function.

To the Editor: We read with interest the contribution entitled, “The structural and functional diversification of the Toxicofera reptile venom system” by B. G. Fry and colleagues. This review article recounts the previous contributions of the authors, presents the authors’ views of the terminology regarding “venom” and Duvernoy’s glands as well as the relevant application of the authors’ interpretations of their phylogenetically based data. The paper also includes dismissal of our concerns and/or recommended cautions about the premature, broad use of the term “venomous” when not supported by evidence of function. In their paper, the authors included some of our published views in total, while others were only partly stated and taken out of context, not mentioned, or in our view, misinterpreted. While refutation of the views presented in the paper requires detailed argument, there are some fundamentals that arise from the paper. Firstly, at the centre of contention is the definition of “venom” and, by extension, “venom glands”. Not only is such definition central to the science of toxinology, but it also has important secondary consequences. If an animal is labelled as “venomous” this can affect the way it is considered by society, the restrictions that are placed on it and the study of the animal, and the attitude, at a community level, towards conservation of the animal. As some readers of Toxicon will know, at the governmental level there are increasing restrictions on the movement and study of anything labelled as a toxin or venom, most recently affecting the study of toxins from cone snails. Although the biological/functional definition of “venom” (see ahead) has nothing to do with medical relevance, the conversely inaccurate labelling of an animal as “non-venomous” can carry serious secondary consequences. These are practical considerations providing a meaningful perspective that cannot be ignored. Secondly, there is the concept of science and the scientific method versus speculation or opinion that can become embedded as scientifically accepted “fact”. As “evidence” will always be selective and partial, it is subject to interpretation based on both the views of the investigators and the selective data being considered by them. Due to these considerations, we recognize that the use of the term, “evidence”, including our employment of it here, has inherent limitations that require further information in order to ascertain a complete objective fact or concept. Thirdly, there is the issue of experimental confirmation, the concept that any new discovery, before formal acceptance, should stand the test of independent experimental confirmation, and be reproducible. Consensus will evolve and change over time, as new information and understanding become available and individual scientists exercise their right to argue in favour of a new consensus. However, they should not, in our opinion, declare they have unilaterally developed a new imposed consensus and “abandoned” specific terminology without broad formal acceptance. In our opinion the paper by Fry et al. (2012) is both premature and non-consensual in its attempt to redefine “venom”, based on its origin (i.e. phylogenetics) rather than by function. Many genes are common, in whole or part, across diverse taxa, doubtless reflecting evolutionary origins, but it is the way each organism has utilised the gene product, in a functional sense, that has traditionally determined the definition of venom and what is “venomous”. The limits we place on a definition clearly affect inclusion/exclusion criteria and rightly are the subject of scientific debate and consensus. Without such care in terminology, humans might be defined as “venomous” (see ahead). Here, we present in detail our disagreement with Fry et al. (2012) regarding the definition of “venom” and “venomous”:

Local envenoming by the Western hognose snake (Heterodon nasicus): A case report and review of medically significant Heterodon bites

A case of clinically significant local envenoming resulting from a bite inflicted by a Western hognose snake, Heterodon nasicus, is described. The patient was bitten while offering a juvenile mouse to a captive snake. The snake maintained a grip on the patients arm (left anticubital fossa) for several minutes. The bite resulted in marked edema, ecchymoses, lymphadenopathy, cutaneous signs suggestive of mild cellulitis and blister formation. There were no systemic effects. Recovery was complete after approximately five months. Several documented Heterodon sp. bites with significant clinical effects are reviewed. This common xenodontine colubrid must be considered capable of inflicting medically significant bites. It is currently unclear whether the pathological changes associated with these bites are due to specific Duvernoys secretion components, Type I hypersensitivity or a combination of these. The influence of the feeding response on the severity of clinical effects is considered as is the discrepancy between experimentally verified pharmacological activities of Duvernoys secretions from Heterodon sp. and medical sequelae of documented bites. Although hognose snakes may uncommonly produce medically significant bites, they should not be considered dangerous or venomous. Captive specimens should be handled carefully, particularly when offered food.

Determination of the in vivo antigen-antibody affinity constant from the redistribution of desipramine in rats following administration of a desipramine-specific monoclonal antibody

Quantitative expressions have been derived to determine the affinity constant for the in vivo interaction of an antigen with its elicited monoclonal antibody by analysing the redistribution of antigen following antibody administration. Using this method, the intrinsic binding constant for the interaction of subtoxic doses of DMI in rats with anti-TCA was found to be about two orders of magnitude less than the value obtained in vitro. The disparity is probably due to the presence of endogenous ligands for the antibody.

Differences Between Buccal Gland Secretion and Associated Delivery Systems of “True” Venomous Snakes and “Colubrid” Snakes: Low- Versus High-Pressure Gland Function and Canaliculated Versus Solid Dentition

An unknown number of non-front-fanged colubroid snakes possess Duvernoy’s glands that produce variably toxic secretions released under low pressure (due to a lack of muscular compression of the glands). This is in marked contrast to the highly muscular and thus pressurized glands of front-fanged colubroids such as viperids, elapids, and atractaspidids. While pressurized venom glands release a bolus of stored venom under high pressure (that may exceed 30psi), non-front-fanged colubroids inoculate their secretions into wounds produced by maxillary teeth that may be enlarged, posterior or mid-maxillary, and may or may not be grooved; but unlike those of front-fanged colubroids, they are never canaliculated (e.g., never hollow, or with a lumen). Dispholidus typus (boomslang), and possibly other members of the tribe, Dispholidini, have limited striated muscle insertion into the gland and thus may be considered to have a partially pressurized venom delivery system. Several hypotheses have been considered that attempt to address the evolution of the venom apparatus and the selection for venom delivery systems. The definition and use of the terms “venom,” and “venom gland” require evidence of biological use of the oral secretion in prey subjugation and/or defense. The possible clinical effects of ophidian oral secretions do not constitute criteria for use of the term “venom.” As evidence of biological use of Duvernoy’s secretion becomes established, some of these complex mixtures may be termed “prey-specific venoms” in order to clarify their medical insignificance, while others, such as those of Dispholidus typus, the African twig, bird, or vine snakes, Thelotornis spp., the keel-backs, Rhabdophis spp., and, possibly, the Montpellier or hooded malpolon, Malpolon monspessulanus, have oral secretions that are used biologically and have concomitant medical importance. Others [such as the Puerto Rican racer, Alsophis (Borikenophis) portoricensis] may have secretions that are demonstrably used to subjugate a variety of prey, and also have occasional medical significance (e.g., a reasonable number of documented bites have caused significant local effects). Thus, the term “venom” may be used for the oral secretions of these taxa without any qualification.

Effect of calcium chloride and 4-aminopyridine therapy on desipramine toxicity in rats

BACKGROUND Hypotension is a major contributor to mortality in tricyclic antidepressant overdose. Recent data suggest that tricyclic antidepressants inhibit calcium influx in some tissues. This study addressed the potential role of calcium channel blockade in tricyclic antidepressant-induced hypotension. METHODS Two interventions were studied that have been shown previously to improve blood pressure with calcium channel blocker overdose. CaCl2 and 4-aminopyridine. Anesthetized rats received the tricyclic antidepressant desipramine IP to produce hypotension, QRS prolongation, and bradycardia. Fifteen min later, animals received CaCl2, NaHCO3, or saline. In a second experiment, rats received tricyclic antidepressant desipramine IP followed in 15 min by 4-aminopyridine or saline. RESULTS NaHCO3 briefly (5 min) reversed hypotension and QRS prolongation. CaCl2 and 4-aminopyridine failed to improve blood pressure. The incidence of ventricular arrhythmias (p = 0.004) and seizures (p = 0.03) in the CaCl2 group was higher than the other groups. CONCLUSION The administration of CaCl2 or 4-aminopyridine did not reverse tricyclic antidepressant-induced hypotension in rats. CaCl2 therapy may possibly worsen both cardiovascular and central nervous system toxicity. These findings do not support a role for calcium channel inhibition in the pathogenesis of tricyclic antidepressant-induced hypotension.