Population pharmacokinetics of Pseudechis porphyriacus (red-bellied black snake) venom in snakebite patients.

OBJECTIVES: Understanding the time course of venom exposure in snakebite patients is important for the optimisation of treatment including antivenom dose and timing. We aimed to investigate the pharmacokinetics of red-bellied black snake (RBBS; Pseudechis porphyriacus) venom in envenomed patients. METHODS: Timed venom concentration data were obtained from patients with RBBS envenomation recruited to the Australian Snakebite Project (ASP), including demographics and antivenom treatment. Venom concentrations were measured using an enzyme immunoassay. Data were modelled using NONMEM version 7.3. Uncertainty in venom "dose" was accounted for by arbitrarily fixing the average amount to 1 mg and incorporating between-subject variability on relative bioavailability. A scale parameter for venom clearance was implemented to account for the rapid venom clearance following antivenom dosing. A sensitivity analysis was performed to determine the magnitude of venom clearance amplification. RESULTS: There were 457 venom concentrations in 114 patients (median age 41, 2-90 y; 80 male). Antivenom was administered to 54 patients a median of 4.2 h post-bite (0.67 to 32 h). A one-compartment model with first-order absorption and elimination provided the best description of the data. The estimated clearance and volume of distribution were 5.21 L/h and 39.9 L, respectively. The calculated elimination half-life of P. porphyriacus venom from the final pharmacokinetic model was 5.35 ± 0.36 h. The variability in the relative dose of injected venom was 140%. Antivenom administration increased venom clearance by 40-fold. Ten patients showed evidence of a double peak in the absorption profile. CONCLUSION: The information on the exposure time of venom in the body following envenomation will help improve treatment and the timing of antivenom.

Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination.

Biodistribution and kinetic studies of technetium-99m labeled Naja naja karachiensis venom via gamma scintigraphic and SPECT images.

Naja naja karachiensis have been responsible for plentiful deaths in Pakistan. To investigate bio distribution and blood kinetics, venom was labeled with the radiotracer (technetium-99m) by following the method of direct labeling technique. Its maximum labeling percentage was 97.7% (pH 6, 100 µg stannous chloride dihydrate) which was higher than some other reported venom. Radio labeled venom was stable for more than 4 hours both in vivo (96%) and in vitro (serum 94.1%, saline 94.3%) experimentations. Intravenous doses of venom (250 µg, 0.5 mCi) were found to be evenly distributed (having R/L ratio=1.0) in all parts of sacrificed rabbits. Kidneys (53.75% activity/g) and urinary bladder (23.70% activity/g) were found with the copious quantity of injected dose of venom. Rest of all other organs was found with subsequent remaining dose of venom. Among them, lungs (14.2% activity/g), liver (4.32% activity/g), bones (1.38% activity/g), heart (0.8% activity/g), blood (0.56% activity/g), skin (0.45% activity/g), intestines (0.35% activity/g), skeleton muscles (0.3% activity/g), brain (0.14% activity/g) and stomach (0.05% activity/g) are included. After 24 hours of injection, poisoned blood of rabbits was almost cleared from venom. Gamma scintigraphic images (up to 2 hours) along with bio distribution suggest that kidneys are main organs of excretion in rabbits. Elimination started immediately after administration of venom however, possible sites for metabolism of venom are liver and lungs. More accumulation of venom in heart compared to brain suggests its higher affinity (thus possible higher toxicity) to cardiac muscles as compared to brain tissues.

Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits.

BACKGROUND: The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. PRINCIPAL FINDINGS: The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole venom (Tmax=2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m.=81.5%) compared to that of the phospholipase A2 (Fi.m.=68.6%) or cardiotoxin (Fi.m.=45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. CONCLUSION/SIGNIFICANCE: Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.

Pharmacokinetics and pharmacodynamics of the myotoxic venom of Pseudechis australis (mulga snake) in the anesthetised rat.

CONTEXT: Myotoxicity is a common clinical effect of snake envenoming and results from either local or systemic myotoxins in snake venoms. Although numerous myotoxins have been isolated from snake venoms, there has been limited study on the relationship between the time course of venom concentrations (pharmacokinetics) and the time course of muscle injury measured as a rise in creatine kinase (CK) (pharmacodynamics). OBJECTIVE: The aim of this study was to develop an in vivo model of myotoxicity to investigate the time course of myotoxicity and the effect of antivenom. MATERIALS AND METHODS: Anesthetised rats were administered Pseudechis australis (mulga snake) venom either through i.v., i.m. or s.d. route, including a range of doses (5-100 µg/kg). Serial blood samples were collected for measurement of venom using enzyme immunoassay and measurement of CK and creatinine. Antivenom was administered before, 1 and 6 h after venom administration to investigate its effect on muscle injury. Plots of venom and CK versus time were made and the area under the curve (AUC) was calculated. RESULTS: There was a significant dose-dependent increase in CK concentration after administration of P. australis venom, which was greatest for i.v. administration. Timed measurement of venom concentrations showed a rapid absorption through s.d. and i.m. routes and a delayed rise in CK concentrations following any route. Antivenom prevented myotoxicity shown by a decrease in the CK AUC, which was most effective if given earliest. There was a rise in creatinine following i.v. venom administration. CONCLUSION: The study shows the delayed relationship between venom absorption and the rise in CK, consistent with the delayed onset of myotoxicity in human envenoming. Antivenom prevented myotoxicity more effectively if given earlier.

Toxicokinetics of Naja sputatrix (Javan spitting cobra) venom following intramuscular and intravenous administrations of the venom into rabbits.

Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms. The toxicokinetics of Naja sputatrix (Javan spitting cobra) venom was investigated following intravenous and intramuscular injections of the venom into rabbits using double-sandwich ELISA. The toxicokinetics of the venom injected intravenously fitted a two-compartment model. When the venom was injected intramuscularly, the serum concentration-time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were not significantly different (p > 0.05) from that of the venom injected intravenously. The systemic bioavailability of the venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some cobra venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn't significantly alter the toxicokinetics of N. sputatrix venom although it significantly reduces the systemic bioavailability of the venom.

In-vivo evaluation of an in situ polymer precipitation delivery system for a novel natriuretic peptide.

This study reports on the release of a novel natriuretic peptide, CD-NP, from an in situ polymer precipitation delivery system. Following extensive screening of in-vitro release profiles, an in-vivo evaluation of the efficacy of the delivery system was carried out in Wistar rats. Gel injection was performed subcutaneously on the back of the rats. A secondary messenger, cyclic Guanosine 3'5' Monophosphate (cGMP), was tested for verification of CD-NP bioactivity, in addition to direct measurements of CD-NP levels in plasma and urine using a radio-immuno assay. Plasma evaluation showed an elevated level of CD-NP over 3 weeks' duration. Unexpectedly, plasma cGMP level followed a decreasing trend over the same duration despite high CD-NP level. Loss of drug bioactivity was ruled out as a high level of CD-NP and cGMP excretion was observed in the treatment group as compared to baseline readings. This unexpected low-plasma cGMP levels and high-urinary cGMP excretion suggest that there might be other compensatory responses to regulation of the CDNP bioactivity as a result of the high drug dosing. The results stress the importance of assessing the overall bioactivity of released drug (in-vivo) concurrently in addition to measuring its concentrations, to determine the correct release profile.

Lymphatic route of transport and pharmacokinetics of Micrurus fulvius (coral snake) venom in sheep.

The contribution of the lymphatic system to the absorption and systemic bioavailability of Micrurus fulvius venom after subcutaneous (SC) administration was assessed using a central lymph-cannulated sheep model. Micrurus fulvius venom was administered either by intravenous bolus (IV) or subcutaneous injection (SC) in 12 sheep with and without thoracic duct cannulation and drainage. Venom concentration in serum and lymph was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) in samples collected over a 6-hour period and in tissues harvested at the end of the experiment. Pharmacokinetic parameters were determined by a non-compartmental analysis. In the lymphatic cannulated group, over the 6 hours after the venom was administered, 69% of administered dose was accounted for in blood (45%) and lymph (25%). Negligible levels of venom were detected in organs and urine implying that the steady state observed after SC administration is maintained by a slow absorption process. Comparison of kinetics of the thoracic duct cannulated and non-cannulated groups showed that lymphatic absorption contributed in an important way to maintenance of this steady state. These results show that the limiting process in the pharmacokinetics of Micrurus fulvius venom following SC administration is absorption, and that the lymphatic system plays a key role in this process.

Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia.

Antibodies that immunoprecipitate (125)I-alpha-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan's syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with (125)I-alpha-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan's syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients.

A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica.

Short- and long-chain alpha-neurotoxins from snake venoms are potent blockers of nicotinic acetylcholine receptors (nAChRs). Short alpha-neurotoxins consist of 60-62 amino acid residues and include 4 disulfide bridges, whereas long alpha-neurotoxins have 66-75 residues and 5 disulfides. The spatial structure of these toxins is built by three loops, I-III "fingers," confined by four disulfide bridges; the fifth disulfide of long-chain alpha-neurotoxins is situated close to the tip of central loop II. An accurate knowledge of the mode of alpha-neurotoxin-nAChR interaction is important for rational design of new nAChR agonists and antagonists for medical purposes. Ideas on the topography of toxin-nAChR complexes were based until recently on nAChR interactions with selectively labeled alpha-neurotoxins, mutations in toxins, nAChR, or both. Recently, crystal structures have been solved for the Torpedo marmorata nAChR (4A[Unwin, 2005]) and for the acetylcholine-binding protein (AChBP) complexed with mollusk alpha-conotoxin (2.4 A[Celie et al., 2005]) or alpha-cobratoxin, long-chain alpha-neurotoxin (4 A [Bourne et al., 2005]). However, there were no angstrom-resolution models for complexes of short-chain alpha-neurotoxins. Here, we report the model of the Torpedo californica nAChR extracellular domain complexed to a short-chain alpha-neurotoxin II (NTII) from Naja oxiana cobra venom.

Novel snake venom ligand dendroaspis natriuretic peptide is selective for natriuretic peptide receptor-A in human heart: downregulation of natriuretic peptide receptor-A in heart failure.

The natriuretic peptides are considered to be cardioprotective; however, their receptors have not been identified in human myocardium using radiolabeled analogs. Dendroaspis natriuretic peptide (DNP) has been recently identified as a new member of this peptide family and is thought to be less susceptible to enzymatic degradation. Therefore, we have developed the novel radiolabeled analog [125I]-DNP and used this to localize high-affinity (K(D)=0.2 nmol/L), saturable, specific binding sites in adult human heart (n=6) and coronary artery (n=8). In competition binding experiments, atrial natriuretic peptide and brain type natriuretic peptide had greater affinity for [125I]-DNP binding sites than C-type natriuretic peptide and the natriuretic peptide receptor (NPR)-C ligand, cANF. This rank order of potency suggested binding of [125I]-DNP was specific to NPR-A. Messenger RNA encoding NPR-A was identified in left ventricle and coronary artery smooth muscle, and expression was confirmed by immunocytochemical studies at the protein level. In addition, fluorescence dual labeling immunocytochemistry localized NPR-A protein to cardiomyocytes, endocardial endothelial cells, and smooth muscle of intramyocardial vessels. Importantly, we demonstrated a significant downregulation in the density of NPR-A in heart and coronary artery of patients with ischemic heart disease that may explain, in part, the attenuated natriuretic peptide response reported in this patient group.

Dendroaspis natriuretic peptide system and its paracrine function in rat colon.

Dendroaspis natriuretic peptide (DNP), a 38-amino-acid peptide, was isolated from the venom of Green Mamba. It has structural and functional similarities to other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP system is present in the rat colon and to define its biological functions. The serial dilution curve of extracts of colonic tissues was parallel to the standard curve of DNP and a major peak of molecular profile by HPLC was synthetic DNP. The concentration of DNP was 0.5 +/- 0.04 ng/g of colonic tissues. DNP as well as atrial natriuretic peptide and C-type natriuretic peptide caused dose-dependent increases in cGMP production in the purified membrane of colonic tissues. Three types of natriuretic peptide receptor mRNAs were detected using semi-quantitative RT-PCR. Functionally, synthetic DNP inhibited the spontaneous contraction of rat colonic circular muscle in a concentration-dependent manner. The potency appeared to be at least 10 times greater than that of CNP. Furthermore, DNP inhibited carbachol-induced muscle contraction, suggesting that it also can modulate the nerve regulation of colonic motility. This study demonstrates the presence of DNP system in rat colon and its function as a local regulator of colonic motility.

Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2.

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.

Renal actions of synthetic dendroaspis natriuretic peptide.

BACKGROUND: Dendroaspis natriuretic peptide (DNP), recently isolated from the venom of the green Mamba snake Dendroaspis angusticeps, is a 38 amino acid peptide containing a 17 amino acid disulfide ring structure similar to that of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). DNP-like immunoreactivity (DNP-LI) was reported to be present in human plasma and atrial myocardium and to be elevated in human congestive heart failure. Although previously named DNP, it remains unknown if DNP is natriuretic or if is it present in canine plasma, urine, and atrial myocardium. METHOD: Studies were performed in vivo in anesthetized dogs (N = 6) using intravenous infusion of synthetic DNP at 10 and 50 ng/kg/min. Employing a sensitive and specific radioimmunoassay for DNP, the presence of DNP-like peptide was assessed in the canine plasma and urine before, during, and following the administration of exogenous synthetic DNP. Additionally, we performed immunohistochemical studies using the indirect immunoperoxidase method with polyclonal DNP antiserum in normal atrial myocardium (N = 10). Atrial concentrations of DNP-LI were also assessed. RESULTS: We report that DNP is markedly natriuretic and diuretic, which, like ANP and BNP, is associated with the increase in urinary and plasma cGMP. DNP-like peptide is also detected in canine plasma, urine, and atrial myocardium. CONCLUSION: These studies establish that DNP is a potent natriuretic and diuretic peptide with tubular actions linked to cGMP and that DNP may play a physiological role in the regulation of sodium excretion.

Pharmacokinetics of 125I-labelled Walterinnesia aegyptia venom and its distribution of the venom and its toxin versus slow absorption and distribution of IGG, F(AB')2 and F(AB) of the antivenin.

A three-compartment open pharmacokinetic model best fitted the data obtained following the i.v. injection of the venom, toxin and the immunoglobulin fractions into either rabbits or mice. The venom and toxin, however, possessed pharmacokinetic characteristics that were significantly different from the immunoglobulin fractions. The venom and toxin had very highly significantly greater disposition rate constants to the shallow and deep tissue compartments and overall elimination rate constant from the central compartment than any of the immunoglobulin fractions. This was reflected in other pharmacokinetic parameters, including highly significantly smaller areas under the curve (AUC) and highly significantly greater volumes of the central compartment (Vc), shallow tissue compartment (Vt shallow), deep tissue compartment (Vt deep) and total body clearance (TBC). In rabbits, F(ab')2 possessed the fastest disposition rate constants and the shortest distribution half-lives, while Fab showed the slowest disposition rate constants and the longest distribution half-lives. The same picture occurred in mice except that the values for Fab were between those of F(ab')2 and IgG. The time needed by the venom and toxin to reach maximum tissue concentration (tmax) ranged between 7 and 15 min and 60 and 180 min for the shallow and deep tissue compartments, respectively. The immunoglobulin fractions required 8-26-fold these times to attain tmax; F(ab')2 was the fastest to achieve its maximal concentration. Following i.m. injection, very fast absorption of venom and toxin took place, with the toxin reaching tmax within 5-20 min and 90% of the injected dose absorbed within 60 min. The bioavailability factor (F) was 0.82 and 0.88 for the venom and toxin, respectively. Fab had an F-value of 0.36 and required 4.3 and 47.4-fold the time taken by the venom and toxin to achieve tmax. The calculated values of F for F(ab')2 and IgG were 0.25 and 0.26, respectively. In the physiologically based pharmacokinetics (PBPK), the venom and toxin reached tmax in the different organs studied very rapidly while the immunoglobulin fractions required several-fold this time to attain tmax. F(ab')2 possessed the highest CPmax, the smallest AUC and the shortest t1/2 beta in the different tissues; Fab had values between F(ab)2 and IgG. It is concluded that F(ab')2 possesses pharmacokinetic characteristics that render it most suitable for use in serotherapy of snake and scorpion envenoming. It should be injected i.v. in doses higher than calculated neutralizing doses to compensate for the slow rate of distribution. Because of slow and incomplete absorption, the i.m. injection of the immunoglobulin fractions would be of little value in serotherapy.

Tissue targeting and plasma clearance of cobra venom factor in mice.

The tissue targeting and rate of clearance of cobra venom factor (CVF) from the circulation was studied in mice by intravenous or intraperitoneal injection of radioiodinated CVF. In both modes of administrations, CVF was targeted mainly to liver. CVF injected directly into the blood was cleared from the circulation with a plasma half life of about 10 h, whereas CVF injected into the peritoneal cavity was slowly absorbed into the blood stream reaching a maximum level at approximately 6 h, and it was then cleared from the circulation with a plasma half life of about 18 h. The rate of plasma clearance of CVF was markedly decreased upon removal of the terminal alpha-galactosyl residues of the oligosaccharide chains; the plasma half lives for intravenously and intraperitoneally administered de-alpha-galactosylated CVF were approximately 5 and approximately 10 h, respectively. However, the clearance rate was not affected by complete deglycosylation using N-glycanase or by chemical modification of the terminal galactosyl residues. Together, these data demonstrate that the terminal alpha-galactosyl residues of CVF mask the Lewis X-dependent uptake of CVF by liver.

Localization of M1 muscarinic receptors in rat brain using selective muscarinic toxin-1.

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M1 receptors in homogenates of rat cerebral cortex. Iodination of the toxin, 125I-muscarinic toxin-1 (125I-MT-1), renders the toxin selective for M1 muscarinic receptors. Quantitative measurement of 125I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of 125I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.

A three-compartment open pharmacokinetic model can explain variable toxicities of cobra venoms and their alpha toxins.

The pharmacokinetic profiles of labelled Naja melanoleuca, Naja nivea, Naja nigricollis and Naja haje venoms and their alpha neurotoxins were determined following rapid i.v. injection into rabbits. The data obtained fitted a triexponential equation characteristic of a three-compartment open pharmacokinetic model comprising a central compartment 'blood', a rapidly equilibrating 'shallow' tissue compartment and a slowly equilibrating 'deep' tissue compartment. The distribution half-lives for the shallow compartment ranged from 3.2 to 5 min, reflecting the rapid uptake of venoms and toxins compared with 22-47 min for the deep tissue compartment denoting much slower uptake. The overall elimination half-lives, t1/2 beta, ranged from 15 to 29 hr, indicating a slow body elimination. Peak tissue concentration was reached within 15-20 min in the shallow tissue compartment. The corresponding values for the deep tissue compartment were 120 min for N. melanoleuca and N. nigricollis venoms and their toxins and 240 min for N. nivea and N. haje venoms and their toxins. Steady-state distribution between the shallow tissue compartment and the blood gave values of 0.50 and 0.92 (N. melanoleuca), 1.64 and 1.05 (N. nivea), 0.78 and 0.92 (N. nigricollis) and 1.70 and 1.03 (N. haje) for the venoms and their toxins, respectively. The corresponding values for the deep tissue compartment gave ratios of 3.31 and 3.44 (N. melanoleuca), 2.99 and 1.68 (N. nivea), 3.74 and 3.79 (N. nigricollis) and 1.39 and 2.46 (N. haje) for the venoms and their toxins, respectively. Ratios lower than unity indicate lower venom and toxin concentrations in the tissues than in the blood, while larger ratios denote higher tissue concentrations. The values thus reflect a higher affinity of the venoms and their toxins for the central than the shallow tissue compartment and for the deep tissue than the central compartment. The sites of action of the venoms seem to be located in the deep tissue compartment since most of the pharmacological, biochemical and electrocardiographic effects of the venoms started 30-60 min after i.v. injection. The mean residence time in the body, MRTb, ranged from 20.8 to 51.8 hr, which correlated well with the long duration of the pharmacological and biochemical effects induced by the venoms. The tissue distribution of the venoms and toxins was similar, with the highest uptake being in the kidneys, followed by the stomach, lungs, liver, spleen, intestine, heart and diaphragm. Very high radioactivity was found in the stomach contents, which reached values higher than the kidneys. Some of the biochemical markers were significantly changed by one or more venoms but the grouped parameters did not reflect significant changes in cardiac, renal, hepatic or electrolyte profiles as a function of time. It is concluded that antivenom, even if injected several hours after a cobra bite, is still capable of neutralizing the slowly eliminating venom. To speed up neutralization of the venom effects, doses of antivenom higher than the calculated in vitro neutralizing dose ought to be injected to compensate for the slow rate of transfer of antivenom to the tissues.

[Distribution of 131I-cytotoxin 14 from Naja naja atra venom in rats].

Cytotoxin 14 (CT14) from Naja naja atra venom was labelled with 131I by chloramine-T method and its tissue distribution was studied in rats. The highest concentration of the cytotoxin was found in kidney, 5979 dpm per mg weight, 14 times more than that of the control animals, at 0.5 h after i.v. injection and high concentrations were found in liver, spleen, pancreas, and adrenal. CT14 was also found in brain at 2 h after injection, 50 dpm per mg weight, 3 times more than that of the control.

Pharmacokinetics of cytotoxin from Chinese cobra (Naja naja atra) venom.

The pharmacokinetics of cytotoxin from Chinese cobra (Naja naja atra) venom was studied in rabbits after i.v. and i.m. injection of 0.2 and 0.5 mg.kg-1, respectively. The plasma levels of the cytotoxin were analysed by a biotin-avidin enzyme-linked immunosorbent assay. The plasma concentration-time course after i.v. administration fitted a two-compartment open model. The half-life (mean +/- S.D.) of the alpha-phase was 5.8 +/- 0.6 min and that of the beta-phase 3.5 +/- 0.2 hr. Apparent volume of distribution was 1.7 +/- 0.3 litres.kg-1, and clearance was 5.6 +/- 1.4 ml.min-1. A rapid absorption was observed after i.m. injection with peak plasma level of 260 +/- 90 ng.ml-1 reached within 13.6 +/- 2.4 min. The absorption rate constant was 0.16 +/- 0.03 ml-1. The area under the plasma concentration-time curve was 33 +/- 15 micrograms.min.ml-1.