MODELLING TITYUS SCORPION VENOM AND ANTIVENOM

PHARMACOKINETICS. EVIDENCE OF ACTIVE

IMMUNOGLOBULIN G's F(ab')2 EXTRUSION MECHANISM

FROM BLOOD TO TISSUES.

Authors: C. Sevcik1, G. D'Suze1, P. Díaz1, V. Salazar2, C. Hidalgo1, H. Azpúrua3, N.

Bracho3.

Institutional affiliations: 1Laboratory of Cellular Neuropharmacology, 2Histology

Service from the Centro de Biofísica y Bioquímica and 3Unit of Experimental

Surgery, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado

21827, Caracas 1020A, Venezuela.

Corresponding Author: Dr. Carlos Sevcik, CBB IVIC, Apartado 21827, Caracas,

1020A, Venezuela

Phone: +58 212 504 1399

Mobile Phone: +58 412 931 9162

Fax: +58 212 504 1764

e-Mail: csevcik@ivic.ve

Running Title: Scorpion venom and ativenom pharmacokinetics.

Key words: Scorpion venom, pharmacokinetics, immunoglobulin transport, IgG, F

(ab')2, antivenom.

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Abstract

Modelling Tityus scorpion venom and antivenom pharmacokinetics.

Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from

blood to tissues. We measured pharmacokinetic parameters for T. discrepans

venom in rams. 40, 75 or 100 µg/kg venom were injected subcutaneously in the

inner side of the thigh. Plasma venom content (venenemia) was determined by

enzyme-linked immunosorbent assay (ELISA) from 0 to 300 min after injecting

venom. Venenemia was fit to a 3 compartment model (inoculation site, plasma and

extra vascular extracellular space), it was assumed that the venom may also be

irreversibly removed from plasma. Calculated time course of venom content shows

that at any time no more that 30% of the venom is present in plasma. Venenemia

peaks at 1h and decays afterwards. Fluorescently labelled antivenom [horse anti-

Tityus F(ab')2 or fraction antigen binging, immuglobulin without Fc chain covalently

bound to fluorescine or fluorescamine] pharmacokinetics was determined.

Although F(ab')2 molecular weight is ≥10 times bigger that toxin's, the rate of

outflow of F(ab')2 from blood to tissues was ≈4 times faster than the venom's

outflow. Venom content in the injection site decays exponentially for >6 h, this

prediction was confirmed immunohistochemically. Only ≈5% of the venom is

eliminated in 10 h; ≈80% of the venom is in the tissues after 2 h and remains

there for >10 h.

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INTRODUCTION

Knowledge of a drug's pharmacokinetics is crucial to understand its action.

Several models have been used (rabbits, rats, mice, guinea pigs) to study venom

pharmacokinetics in scorpions such as Androctonus amoreuxi, Leiurus

quinquestriatus, Buthotus judaïcus, Androctonus crassicauda (Ismail et al., 1980,

1988, 1992, 1994) and Centruroides limpidus limpidus (Calderón-Aranda et al.,

1999). These studies were devised to measure distribution and elimination of 125I

labelled venom injected to experimental animals. Yet, this technique is indirect and

does not distinguish between 125I bound to toxic and non toxic venom components,

and may produce inaccurate results if the isotope is interchanged with proteins of

the host. Thus, the isotope measured in blood may be misleading (Krifi et al.,

2001).

A more direct approach has been used by Santana et al. (1996) and Krifi et al.

(2001) who employed a sandwich type ELISA to measure venom concentration of

Tityus serrulatus and Buthus occitanus tunetanus respectively, obtaining more

accurate values for pharmacokinetic parameters. The results of these authors were

coincident in showing a biphasic time course for both species with a rising phase

followed by a decay, with a maximum concentration between 30 and 60 min and

obtained pharmacokinetic parameters indicative of a fast absorption and high

distribution in tissues.

In this communication we describe experiments on pharmacokinetics of the

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Tityus discrepans scorpion venom in rams using a modification of the high

sensitivity ELISA method of developed in our laboratory for Tityus venom (D'Suze

et al., 2003). Horse anti-Tityus F(ab')2 (fraction antigen binging, immuglobulin

without Fc chain) was covalently bound to either fluorescine or fluorescamine and

its pharmacokinetics also measured. Although the molecular weight of F(ab')2 is

≥10 times bigger that the toxins, the rate of outflow of F(ab')2 from blood to tissues

was ≈4 times faster than the venom's outflow, suggesting that the antivenom is

actively transported from blood to tissues. Using the experimentally determined

kinetic constants we modelled the distribution of venom and F(ab')2 in the body

and showed that plasma compartment is a transient pathway from the

envenoming site to the target organs, therefore the venenemia underestimates the

amount of venom received; this underestimation grows with envenoming time.

Also, results where F(ab')2 therapy is introduced in the model, remark the

importance of the early use of anti venom, ideally during the first hour of

envenoming.

Materials and Methods

Experimental methods

T. discrepans scorpions were anaesthetised with CO2 and milked monthly for

venom by electrical stimulation. The venom was dissolved in bidistilled water and

centrifuged at 15,000 g for 15 min. The supernatant was freeze-dried and stored at

-80°C until used. Specific anti-T. discrepans F(ab')2 were commercially obtained

horse anti-venom (Suero Anti-escorpiónicoTM, Centro de Biotecnología UCV,

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Caracas, Venezuela).

Pharmacokinetic parameters were determined for the venom of Tityus

discrepans in rams (≈40 kg). The anaesthesia was induced with propofol (4 mg/kg)

and was maintained with 1 to 2 % isofluorane in 100% O2; hydration was

maintained with a continuous intravenous (iv) infusion of 0.9% NaCl saline at a rate

of 1.6 ml/h/kg plus 8 ml boli of heparinised saline after drawing each blood sample

to replace the plasma volume and to keep the catheter permeable during the

whole procedure. After 5 h of experiment the animals were sacrificed by an

overdose of thiopentone (20 mg/kg iv). The venom was injected subcutaneously in

the inner face of the thigh in doses of either 40, 75 or 100 µg/kg. After

administering the venom (time = 0), 8 ml blood samples were draw at 2 min

intervals during the first 10 min, then at 5 min intervals during the next 20 min and

finally each 10 min up to the end of the experiment. Plasma was separated from

whole blood and kept at -80°C until used.

The concentration of active venom in plasma (called venenemia from know

on) was determined with a modification of the specific sandwich ELISA published

by D'Suze et al. (2003). Maxisorp (Nunc Inc., USA) plates were coated in a wet

chamber for 12 h at 4°C with 10 µg/ml anti-T. discrepans F(ab')2, in 100 mM

carbonate/bicarbonate (pH 9.5) (100 µl/well). The plates were then washed with

washing buffer (50 mM Tris/HCl, pH 8) and the remaining active sites in the wells

were blocked with 3 % skim cow milk (percentages of skim milk always refer to

powdered milk weight in this paper) in washing buffer (100 µl/well, 1 h at 37°C).

After washing 100 µl/well control plasma with a known venom concentration

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spanning from 0.45 to 166 ng/ml were used as calibration curve. Otherwise 100

µl/well experimental ram plasma was used; the calibration and experimental wells

were incubated 1 h at 37oC. After washing the plates were incubated 1 h at 37oC

with 125 ng/ml anti-Tityus rabbit immunoglobulin G (IgG) produced in our

laboratory; the plates were washed and incubated again (1 h at 37oC, 100 µl/well)

with anti-rabbit IgG conjugated with horse radish peroxidase (HRP) (Zymed

California, EEUU) diluted 1:5000 in washing buffer. The plates were then washed

and developed with ABTS [2,2'-azino-bis(3-ethylbenziazoline-6-sulfonic acid),

Zymed, CA U.S.A.] diluted 1:100 with washing buffer (100 µl/well). The reaction was

halted with 2 N FH (25 µl/well) and the absorbance of the wells was measured at

405 nm with a Multiskan Spectrum spectrophotometer (ThermoLife Sciences,

Basingstoke, UK). This method was found to be better than the one previously

published (D'Suze et al., 2003) since it produces extremely linear calibration curves

with very high correlation.

Fluorescent labeling of antivenom.

The kinetics of F(ab')2 in rams was measured using fluorescent anti venom

labeled with fluorescin or fluorescamine. labeling with fluorescin was done using 2

ampoules of anti venom (10 ml) which were mixed with 4.5 ml of 50 mM sodium

borate buffer pH 9.6 and 0.5 ml of a solution 4% fluorescin in acetone were added

under vigorous agitation in a vortex stirrer. The tube was then completely covered

with aluminum foil and the mixture was left at room temperature (≈22oC) for 6 h

and then at 4oC for 12 to 36 h. The mixture was then washed by ultrafitration with

150 ml of 50 mM sodium borate buffer pH 8 through an AMICON-Millipore

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membrane (PBDK type, 50 kDa cut-off, 25 mm diameter) under nitrogen pressure

(4 bars) at 10oC in darkness. The labelled material was thus reduced to between 5

and 6 ml and washing buffer was added to make 10 ml. The final product was kept

in a small balloon wrapped in aluminum foil and stored at 4oC for not more than

24 h before using it.

Labeling with fluorescamine (Udenfriend et al., 1972; Böhlen et al., 1973; De

Bernardo et al., 1974; Stein et al., 1973; Stein et al., 1974) was done by mixing,

under vigorous vortex stirring, 2 ampoules of antivenom with 2 ml of 250 mM pH 9

borate buffer, still under vigorous stirring 4 ml of fluorescamine in acetone (30

mg/100 ml) were added to the mixture which was stored at 5oC in darkness for ≈12

h prior to using. Vigorous stirring is of fundamental importance since the reaction

of the fluorescamine and amines occurs in milliseconds and the unreacted

fluorophore hydrolyses spontaneously and completely in a few seconds (De

Bernardo et al., 1974; Stein et al., 1974). Ten of the 16 ml of resulting mixture were

injected iv to a ram to determine kinetics, the remaining solution was reserved for

controls in high performance liquid chromatography (HPLC) procedures.

The mechanisms of reaction between the fluorophores and primary amines

is shown in figure 1. The mechanism of reaction of fluorescin is based on chemical

information from Nesmeyanov and Nesmeyanov (1981) and the reaction of

fluorescamine is based on De Bernardo et al., (1974) and Stein et al., (1974).

High Performance Liquid Chromatography Methods

Both the original anti venom as well as the fluorescin or fluorescamine

labeled F(ab')2 where processed with a Shimadzu LC 6B (Shimadzu Corp., Kyoto,

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Japan) HPLC system using molecular exclusion Protein-Pak 300SW HPLC (Millipore

Corporation, Waters Chromatography Division, Milford, MA, USA) column eluting a

rate of 0.5 ml/min with 100 mM Tris (2-amino-2-hydroxymethyl-1-3-propanediol)

buffer with F3C.COOH added to give pH 8. In some instances the eluate was

monitored either measuring its absorbance at 280 nm (Shimadzu SPD-6AV,

ultraviolet-visible absorbance detector). For the kinetics experiments fluorescence

(Shimadzu RF-551, Spectrofluorimetric detector) at 516 nm (exciting at 490 nm)

was measured when labeling with fluorescin, or at 475 nm (exciting at 390 nm)

when labeling with fluorescamine.

Histological methods

Organ fixation was initiated by perfusion with 10% w/v formaldehyde buffered

with 1/15 M phosphate buffer pH 7.6 of the sacrificed whole animal; the fixation

was completed by immersion of the tissues in the same fixative and Bouin´s fluid

for 24 h at room temperature. The material was dehydrated with ethanol, clarified

with xylene and embedded in Paraplast. Sections between 4 and 8 m, were

dewaxed in xylene, rehydrated with distilled water. After removal from paraffin

with xylene, the endogenous peroxidase was blocked in all tissues with 3% H2O2 for

30 min in methanol between two ethanol changes during rehydration (Birne et al.,

2001; Farioli-Vecchioli et al., 2001). Antigen was reactivated by boiling in 0.01 M

citrate buffer (pH 6) for 10 min (Farioli-Vecchioli et al., 2001). After antigen

reactivation the slides were extensively washed with phosphate buffered saline.

Unspecific binding sites were blocked using phosphate buffered saline (PBS)

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containing 5% skim milk, 0.2% triton X-100 for 1 h at room temperature before

incubation with the primary antibody. Slides were incubated overnight at 4oC with

0.2 mg/ml rabbit polyclonal anti-Tityus IgG diluted 1:1000 in PBS containing 2.5%

skim milk and 0.1% triton X-100. A secondary antibody incubation was made for 1

h at room temperature with peroxidase-complexed mouse anti-rabbit IgG diluted

1:500 in PBS containing 1% mouse normal serum. All the incubation steps were

followed by extensive washing in PBS. Sections were then incubated with 10 µl of a

solution containing 8% NiCl2, 1 ml 0.05% 3-diaminobenzidine tetrahydrochloride,

0.02% H2O2 and 50 mM Tris-HCl, pH 7.6, with all components mixed just before use

(Lobo et al., 2001). Finally the sections were washed with 50 mM Tris-HCl buffer, pH

7.6. This method results in a dark brown colour of immunostained antigens. The

sections were counter stained with Mayer´s haematoxylin (Bradbury Gordon,

1990), mounted in Crystal-Mount (Biomedia Corp., Foster City, CA) and made

permanent with Permount (Fisher Scientific, Fair Lawn, NJ).

Numerical methods

The following simultaneous differential equations, were programmed in

Fortran 77 and were fit to the data using the ADAPT II

Pharmacokinetic/Pharmacodynamic Analysis Software (D'Argenio and Schumitzky,

1997) to obtain empirical values of the kinetic constants:

d Mp

d t=ka⋅Minockep⋅Mev−kpekel ⋅Cp⋅Vp

d Minoc

d t=−ka⋅Minoc

d Mev

d t=kpe⋅Mp−kep⋅Mev

[1]

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Where the symbols are: ka, rate of diffusion of venom from the injection site to

plasma; kel, rate of elimination of venom from plasma; kpe: diffusion rate constant of

venom from plasma to the extra vascular extracellular space; kep: diffusion rate

constant of venom from the extracellular compartment to plasma; Minoc, mass of

venom in the inoculation site; Mp : mass of venom in plasma; Mev: mass of venom in

the extracellular space; Cp, concentration of venom in plasma; Vp, distribution

volume of venom in plasma.

The Adapt II environment was chosen because it is a time-proved freeware

(for nonprofit research) package which simplifies writing the model equations as

Fortran templates, and saves a lot of programming time. The package implements

the simplex algorithm to fit model to datums (Nelder and Mead, 1965), the LSODA

method to solve general differential equations (Hindmarsh, 1982; Petzold, 1983)

and a matricial exponential method to solve linear differential equations (D'Argenio

et al., 1988). The package also permits plotting the fit between model and datums.

The remaining simulations were carried out by solving numerically the kinetic

equations using the Euler method (Carnahan et al., 1969) in 1.8 ms steps,

programmed in C++ using the g++ GNU (www.gnu.org) compiler with the Anjuta

1.2.1 Integrated Development Environment (www.anjuta.org) and plotting data

with OpenOffice.org 1.1 Calc (www.openoffice.org) all under Fedora Core Linux

Release 1.

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Additional definitions

We will use the term "fabotherapeutic", similar to the recently coined Spanish

"faboterapéutico" (Anonymous, 2002) in reference to the antivenom used, meaning

an antitoxin made of F(ab') or F(ab')2. In the model a “milligram F(ab')2 represents

the amount required to neutralise a milligram of venom. This is due to our

conviction that it is intuitively more informative that moles or milligrams of

fabotherapeutic by itself. Since neither the fabotherapeutic nor the venom are

composed of a single well defined molecule, using precise units are only delusions.

In the case of the anti-Tityus fabotherapeutic used in Venezuela, 1 ampoule of

fabotherapeutic neutralises 1 mg (actually 1.1 mg at the time of manufacture) of

T. discrepans venom. Thus from now on we will express the amounts of

fabotherapeutic used interchangeably as mg of venom neutralised or as ampoules.

Actually, 1 ampoule of antivenom contains 181 mg protein determined by

measuring absorbance at 280 nm.

Statistical procedures

The data were processed using nonparametric statistical procedures.

Data are presented as medians and their 95% confidence interval calculated

with the procedure of Hodges and Lehmann. Statistical significance of

differences was decided with Mann-Whitney (Wilcoxon) test. Linear

regressions were carried out with the Theil procedure. See Hollander and

Wolfe (1973) for all details of nonparametric methods used. Differences

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between treatments were considered significant if the probability that the

null hypothesis was true was ≤0.05 (P≤0.05, two tails).

Results

Determining Pharmacokinetic Constants for Venom

The top of figure 2 presents a typical fit between the model represented by

equation set [1] and the plasmatic concentration of venom in a ram. Table 1

presents the kinetic constants obtained when equation set [1] was fitted to data of

5 rams as indicated in methods. In addition to the kinetic constants described in

methods, table 1 presents the venom distribution volumes in plasma and in the

extra vascular extracellular space.

Using the model to make inferences

Since for any of the masses in equation set [1] we have that M=V·C; it is trivial

to transform this set into the following system in terms of distribution volumes and

concentrations:

dCp

d t=

ka⋅Minockep⋅Vev⋅Cev

Vp

−kpekel ⋅Cp

d Minoc

d t=−ka⋅Minoc

dCev

d t=

kpe⋅Vp⋅Cp

Vev

−kep⋅Cev

[2]

where the mass in the inoculation site was preserved due to the lack of

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information on the distribution volume on this compartment. Here: Cev,

concentration of venom in the extra vascular extracellular space; Cp, concentration

of venom in plasma; Vev, distribution volume of venom in the extra vascular

extracellular space. Other symbols as before.

The bottom of figure 2 presents a prediction of Cp and Cev calculated with the

median kinetic parameters in table 1 and with equation set [2]. Both the

experimental and theoretical results in figure 2 are in good agreement between

themselves and with data previously published by other authors (Ismail et al.,

1980, 1988, 1992, 1994; Santana et al., 1996; Calderón-Aranda et al., 1999; Krifi

et al., 2001) in regards with the time course of venenemia.

Prediction for the amount of venom in blood and the targettissues

The data on Cp and Cev in figure 2 and in the literature cited above, are

misleading. Since venom concentration as shown in the figure is higher in plasma

than in the target tissues, the concentration in blood seems to gauge the severity

of envenoming and the damage suffered by the patient. To evidence this falsehood

we modified equation set [1] to obtain the following system of equations

formulated purely in terms of venom masses:

d Mp

d t=ka⋅Minockep⋅Mev−kpekel ⋅Mp

d Minoc

d t=−ka⋅Minoc

d Mev

d t=kpe⋅Mp−kep⋅Mev

[3]

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where all the symbols have the meanings previously described.

Figure 3 presents the results calculated with equation set [3] with 1 mg of

venom administered at time 0. This figure shows that although the time course of

Mp is qualitatively similar to Cp, Mp does not correlate with the time course of Mev at

all. Another unexpected prediction was that Minoc decays exponentially and slowly,

and that by the 5th h an amount of venom still remains at the inoculation site. The

last prediction was confirmed immunohistochemically as it is shown in figure 4,

were the venom was still present in significant amounts at the inoculation site after

5 h.

Determining Pharmacokinetic Constants for anti-T.discrepans F(ab')2

We extended the model to include F(ab')2 assuming that the antivenom does

not diffuse out of the blood compartment, equation set [3] was modified to include

the neutralisation venom-anti venom reaction as follows:

d Mp

d t=ka⋅Minockep⋅Mev−kpekelkf⋅Mfab.p ⋅Mp

d Minoc

d t=−ka⋅Minoc

d Mev

d t=kpe⋅Mp−kep⋅Mev

d Mfab.p

d t=−kf⋅Mp⋅Mfab.p

[4]

where: kf, venom-anti venom reaction rate; Mfab.p, amount of F(ab')2 in plasma. kf

was set as 50,000 min-1.

Figure 5A and 5B presents the predictions of equation set [4]. In both halves

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of the figure 1 mg venom was administered sc at time 0, but 1 ampoule of

fabotherapeutic was administered iv at minute 60 in 5A and 3 ampoules of

fabotherapeutic were given at the same time in 5B. As seen the predictions of the

model are identical for both situations, and show that although free venom in

plasma drops to 0 immediately when F(ab')2 is injected, the amount in the tissues

descends (but exceedingly slowly) afterwards.

Measuring the diffusion of F(ab')2 into and out of the bloodvessels

The exceedingly slow venom rate of descent in tissues and its insensitivity to

fabotherapeutic dose, is not compatible with the clinical experience when scorpion

victims are subject to early fabotherapy, and prompted to consider that F(ab´)2

may diffuse out of blood vessels to neutralise venom in the extravascular space.

To test this hypothesis we assumed that a two compartment model such as:

dMfab.p

dt=k fd.ep⋅Mfab.e−k fd.pe⋅Mfab.p

dMfab.e

dt=k fd.pe⋅Mfab. p−k fd.ep⋅Mfab.e

[5]

describes the F(ab')2 diffusion from plasma to the extracellular extravascular

space, in absence of venom. Here the new symbols mean: kfd.ep, diffusion rate

constant for F(ab')2 from tissues to blood vessels; kfd.pe: diffusion rate constant for F

(ab')2 from blood vessels to tissues; Mfab.e, amount of F(ab')2 in the extracellular

space. Other symbols as defined before.

The kinetic F(ab')2 parameters were determined with fluorescently labeled

15

antitoxin as indicated in Methods. Figure 6 presents experimental points and lines

fitted to them from two experiments, one labeling with fluorescin and another

labeling with fluorescamine. Table II presents the kinetic data for F(ab')2

determined in 3 rams; 2 of them labeling with fluorescin and one labeling with

fluorescamine.

Predictions of fabotherapy with diffusion of F(ab')2 into andout of the blood vessels

Using the parameters in tables 1 and 2 and the following set of simultaneous

differential equations:

dMp

d t=ka⋅Minockep⋅Mev−kpekelkf⋅Mfab.p ⋅Mp

dMinoc

d t=−ka⋅Minoc

dMev

d t=kpe⋅Mp−kepkf⋅Mfab.e ⋅Mev

dMfab.p

d t=kfd.ep⋅Mfab.e−kfd.pekf⋅Mp ⋅Mfab. p

dMfab.e

d t=kfd.pe⋅Mfab.p−kfd.epkf⋅Mev ⋅Mfab.e

[6]

we modeled the effects of different protocols of administering fabotherapy.

Figure 5C presents results obtained under the same conditions used in figure

5A calculated with equation set [6]; venom parameters were as in table 1, and F

(ab')2 parameters as in table 2. The antivenom readily removes the venom from

plasma and extracellular compartments; the remotion from the extracellular

compartment is slightly faster when 3 ampoules of venom are used (Figure 5D)

instead of 1 (Figure 5C).

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Modeling Different Fabotherapeutic Protocols.

Figure 7 presents predictions with equation set [6] for different clinical

situations where antivenom is administered. In all panels 1 mg of venom was

received at time 0, followed by 1 ampoule of antivenom 60 min later in 7A or only

½ ampoule in 7B. When insufficient fabotherapeutic is used, plasmatic venom is

initially completely neutralized, but increases again as venom continues diffusing

from the inoculation site to plasma.

In figure 7C 1 ampoule of anitvenom was administered continuously

and at the same rate (mimicking an iv infusion) from minute 60 through minute

150; as seen the area under the venom in organs curve is much larger than in 7A.

A similar situation is depicted in 7D, where ¼ of ampoule was injected iv at minute

60, and the same amount repeated at minutes 90, 120 and 150. As examples of

late fabotherapy, we also modeled situations where 1 ampoule (7D) or 3 ampoules

(7E) of anitvenom were administered 9 h after receiving venom .

Discussion

We have determined the pharmacokinetic constants for Tityus discrepans

scorpion venom and of its F(ab')2 antivenom in rams. The parameters were used in

a model to describe the distribution and dynamics of both the venom and

antivenom under several treatment modalities. We found an excellent agreement

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between experimentally measured plasmatic venom and antivenom

concentrations with the predictions of the mathematical model used. The model

predicts several unexpected features such as the existence of significant amounts

of venom at the inoculation site 5 h after inoculating the venom. This prediction

was verified experimentally by means of immunohistochemistry (Figure 4).

As shown in table 1 the rate of outflow (kpe) of Tityus venom from plasma is

≈20 times faster that the rate of inflow (kep); this probably results from toxins' high

affinity for tissular receptors. The high affinity (and probably the abundance of

receptors) determines that the distribution volume of venom in the extravascular

space [Ve = 182.8 (117.1, 253.9) lit] is much larger than 4 to 6 lit [10 to 15% of body

weight, Manery, (1954)] as expected for the rams utilised; this overestimation

expresses abundant high affinity extracellular space venom receptors. The venom

distribution in plasma [Vp = 11.2 (2.0, 20.0) lit] is also larger than the expected 2 to

3 lit [5 to 7% of body weight, Manery, (1954)], suggesting that there are high

affinity venom receptors in blood. Much less is known about blood receptors for

Tityus venom, but since the venom has pro- and anticoagulating effects (D'Suze et

al., 2003) they must exist. In contrast with these results, and in spite of the fact

that the outflow and inflow rate constants for F(ab')2 are different, our estimates of

F(ab')2 distribution volumes in plasma and extracellular space are the expected real

volumes of these compartments.

To simplify the solution of the equations, in the calculations using equation

sets [4] and [6] the reaction between F(ab')2 and venom was modeled as

irreversible and with a forward reaction rate kf set to 50,000 min-1. This reaction is

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reversible with a dissociation constant between 10-8 and 10-11 M, and a forward

reaction rate between 3×105 and 4×108 s-1 (Goldsby et al., 2000). Since the

molecular weight of venom components is ≤10 kDa, the value used in the

calculation is ≈8×102 s-1 which is conservative. Thus, the almost instantaneous

neutralization of venom with F(ab')2 is not a model exaggeration.

Given that the molecular weight of F(ab')2 is ≥10 times higher than venom's

toxins, it was surprising to find that the kinetics of fluorescent F(ab')2 extravasation

was, independently of which fluorophore was used, about 4 fold faster that venom

extravasation. The faster antivenom outflow can only be explained in terms of the

existence of some kind of active mechanism of extrusion of F(ab')2 through the

vascular endothelium. It is known that polymeric immunoglobulin A (IgA) and

immunoglobulin M (IgM) are excreted through epithelia (Bradzaeg, 1981; Hühn and

Kraehenbuhl, 1981; Mostov et al., 1980, 1984) by means of a vesicular mechanism.

This mechanism is similar to what has been called potocytosis in mesothelia. In

potocytosis, when specific membrane receptors recognises a molecule, a special

kind of pit called caveola is formed entrapping the ligand and the receptor; the

caveola is then endocyted and may travel to the opposite side of the cell where it

releases the ligand (Mineo and Anderson, 2001). To our knowledge no such

mechanism has yet been demonstrated for IgG. Potocytotic transcytosis has been

shown to occur for albumin, transthyrein, thrombomodulin, several plasma proteins

and water (Mineo and Anderson, 2001; Bodega et al., 2002; Predescu et al., 2002);

the half time (T½) of internalisation by potocytosis is ≈30 min (Mineo and Anderson,

2001). A vesicular mechanism of transcytosis different from potocytosis occurs in

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epithelia and mesothelia, it involves clathrine-coated pits; this mechanism of

endocytosis has a T½ of internalisation of ≈6 min (Mineo and Anderson, 2001). The

value of T½ corresponding to kfd.pe in table 2, 14.2 (9.8, 24.8) min, lies in between

the two values cited, making it difficult to decide at the present, between caveolae

and clathrine-coated pits as a candidates to extravasate IgG. Given that the

antivenom is a fabotherapeutic, if an active extrusion mechanism exists, it does

not require IgG's Fc chain and probably means that IgG is transported as a

monomer. In the experiments using fluorescin labelling, the antivenom was

ultrafiltered for over 24 h to eliminate molecules of ≤50 kDa producing pure F(ab')

2 as it was shown by molecular exclusion HPLC (not shown). The data in tables 1

and 2 shows that ka ≅ kfd.ep; this coincidence suggests that after the antivenom is

extravasated, it returns to plasma via the lymph vessels as it is the case for toxins

in elapid snake venoms and other large molecules (White, 1982).

Venom kinetics modeled using the parameters in table 1 predicts that no more

that 30% of the venom is present in plasma at any time. Venenemia peaks at 1 h

and then decays. Only ≈5% of the venom is eliminated in up to 10 h, ≈80% of the

venom is in the tissues after 2 h and remains there for >10 h when venenemia

becomes negligible. Plasma is a transient pathway from the envenoming site to the

target organs, therefore venenemia underestimates the amount of venom

received; this underestimation grows with envenoming time.

The model is able to predict that with an insufficient dose of antivenom

venenemia may be transiently reduced to nil, but that it will increase again as

venom keeps entering plasma from the inoculation site (figure 7B) and due to the

20

rapid F(ab')2 plasma outflow. The sequence of plasmatic venom neutralization

followed be an increase, repeats it self several times when the antivenom needed

to neutralise the venom is fractioned (figure 7D). When figure 7A is compared to

7C and 7D, it becomes evident at the area under the curve of venom mass in

organs is much larger when antivenom is not administered as a single bolus; a

larger area under the curve of venom in organs means that the poison has a

greater chance to harm. Since most of the worst cases of Tityus poisoning in

Venezuela occur in babies and toddlers, diluting the venom in 50 ml of saline, as it

is sometimes advised, may represent an important and unnecessary blood volume

expansion. Figures 7E and 7F indicate that the area under the curve of venom in

organs grows enormously in cases of late antivenom administration, in spite of the

fast outflow of antivenom from plasma and even if the amount of fabotherapeutic

required tho completely neutralise the venom is raised 3 fold. In Venezuela, most

of the clinically severe cases where antivenom administration is delayed as in

figures 7E and 7F, are fatal or become extremely serious, even with

cerebrovascular complications. The present results suggest that this probably

stems from accumulated damage, which includes a generalised inflammatory

syndrome (D'Suze et al., 2003), rather that from the inability to neutralise tissular

venom. There is no replacement for early fabotherapy.

21

Acknowledgements

This work was partly financed by Laboratorios Silanes SA de CV, Mexico and

by the IVIC-Silanes grant for applied projects No. 2001-31. The assistance of Mr.

Moisés Sandoval and Dr. Tatiana Vzentekova is kindly acknowledged.

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26

Table 1. Kinetic Parameters for Tityus discrepans VenomDetermined in Rams.

Parameter Value T1/2

ka 1.2∙10-2

(0.78, 1.8)∙10-2 min-157.8

(38.5, 88.9) min

kel 1.03∙10-3

(0.56, 3.0)∙10-3 min-1673.0

(231.0, 1237.8) min

kpe 1.5∙10-2

(0.52, 2.7)∙10-2 min-146.2

(25.7, 133.3) min

kep 4.5∙10-4

(2.0, 20.0)∙10-4 min-11540.3

(346.6, 3465.7) min

Vp 11.2(2.0, 20.0) lit

Ve=Vp∙kpe/kep 182.8(117.1, 253.9) lit

Data presented as medians and their 95% confidence interval (between

parentheses) of 5 experiments

27

Table 2. Kinetic Parameters for Venezuelan Fabotherapeutic AgainstTityus discrepans Venom Determined in Rams.

Parameter Value T1/2

kfd.pe 4.9∙10-2

(2.8, 7.1)∙10-2 min-114.2

(9.8, 24.8) min

kfd.ep 1.2∙10-2

(0.7, 1.7)∙10-2 min-157.8

(40.8, 99.0) min

Vfab.p 1.9(0.8, 3.0) lit

Vfab.e=Vfab.p∙kfd.pe/kfd.ep 10.2(5.8, 14.7) lit

Data presented as mean and range (between parentheses) of 3 rams.

28

Legends for the figures

Figure 1- Fluorescent labeling of proteins via primary amine reaction with

fluorescine and fluorescamine. R-NH2 represents the primary amine of a

protein. The mechanism of reaction of fluorescin is based on chemical

information from Nesmeyanov and Nesmeyanov (1981) and the reaction of

fluorescamine is from De Bernardo et al., (1974) and Stein et al., (1974).

Figure 2- Time course of plasmatic venom concentrations (venenemia) in

rams. The top of the figure presents the time course venenemia in one of the

rams studied; squares are experimental values, the line was fitted as

indicated in methods to equation set [1]. The bottom of the panel is the time

course of venom concentration in plasma (Cp, solid line) and in the

extracellular extracellular extravascular space (Cev, dashed line) both

calculated by solving numerically equation set [2] with the median kinetic

constants presented in table 1 assuming that 1 mg of venom was

administered at time = 0.

Figure 3- Time course of venom mass distribution in rams. The figure

presents the time courses predicted with equation set [3] with the median

kinetic constants presented in table 1 assuming that 1 mg of venom was

administered at time = 0. The solid line is the amount of venom in plasma;

29

The short dashed line is the amount of venom in the extra vascular

extracellular space; The dotted line is the amount of venom in the inoculation

site; The dash dot dot line is the amount of venom eliminated from plasma.

Abscissa is the amount of venom in mg; ordinate is time in hours. Other

details in the text of the communication.

Figure 4- Immunohistochemical detection of venominthe inoculation site.

Two sections of the subcutaneous connective tissue where the venom was

injected are presented; the venom appears immunochemically stained in

yellowish brown all over the sections; nuclei are greenish blue. A large

lymphatic vessel (LV) containing venom in the haemolymph is presented in

the top section indicating that venom is still absorbed from the tissue in

significant amounts. In contrast with the lymphatic vessels, the plasma

content of an arteriole in the lower section is clear indicating a much lower

venom concentration. The calibration bar (50 mm) in the lower panel is valid

for both photomicrographs, other details in the text of the communication.

Figure 5- Time course of venom mass distribution in rams when F(ab')2 is

injected iv Panels A and B present the time courses predicted with equation

set [4] assuming that the fabotherapeutic does not diffuse out of the blood

vessels. A- 1 mg of venom was administered at time 0 followed by 1 ampoule

of fabotherapeutic. B- 1 mg of venom was administered at time 0 followed by

3 ampoules of fabotherapeutic. Panels C and D present the time courses

predicted with equation set [5] using the diffusion constant for F(ab')2

30

determined experimentally as shown in figure 4. C- 1 mg of venom was

administered at time 0 followed by 1 ampoule of fabotherapeutic. D- 1 mg of

venom was administered at time 0 followed by 3 ampoules of

fabotherapeutic. In all cases the fabotherapeutic was administered iv 60 min

latter. Other details as in figure 2 and in the text.

Figure 6- Time course of fluorescent F(ab')2 injected iv in rams. The

squares are experimental data, the lines were fitted with equation set [6]

using the kinetic parameters in tables 1 and 2. In the top of the figure is one

experiment where the antivenom was labeled with fluorescine. In the bottom

is another experiment labeling the antivenom with fluorescamine. As may be

appreciated the time course of F(ab')2 in plasma is very similar in both cases.

Other details like in figure 3 and in the text of the communication.

Figure 7. Modelling Different Fabotherapeutic Modalities. The figure present

model predictions for the use of insufficient, slow and late administration of F

(ab')2. In all panels 1 mg of venom is administered at time 0. In panels A

trhough D 1antivenom was administered 60 min after venom's

administration. In panel A the ampoule was given as singe iv bolus. In panel B,

only ½ ampoule was given as a single bolus. In panel C, 1 ampoule is

administered as a continuous iv infusion from minute 60 through minute 150.

In panel D, ¼ of ampoule was injected iv at minute 60, and then again at

minutes 90, 120 and 150. In panel E, 1 ampoule of anitvenom was

administered iv 9 h after venom. In panel F, 3 ampoules were administered iv

31

9 h after venom. Other details like in figures 3 and 6, and in the text of the

communication.

32

Figure 1. Sevcik et al.

33

Figure 2- C. Sevcik et al.

34

0 100 200 300 400 500 6000

10

20

30

Time (min)

Ven

om

Co

nce

ntr

atio

n (

ng

/ml)

Figure 3- C. Sevcik et al.

35

0 2 4 6 8 100.00

0.20

0.40

0.60

0.80

1.00

Time (hr)

Venom

Mass

(m

g)

Figure 4- C. Sevcik et al.

36

Figure 5- Sevcik et al.

37

0 2 4 6 8 10

0.00

0.20

0.40

0.60

0.80

1.00

A

Time (hr)

Venom

Mass

(m

g)

0 2 4 6 8 10

0.00

0.20

0.40

0.60

0.80

1.00

B

Time (hr)

Venom

Mass

(m

g)

0 2 4 6 8 100.00

0.20

0.40

0.60

0.80

1.00

C

Time (hr)

Venom

Mass

(m

g)

0 2 4 6 8 100.00

0.20

0.40

0.60

0.80

1.00

D

Time (hr)

Venom

Mass

(m

g)

Figure 6. Sevcik et al.

38

Figure 7. Sevcik et al.

39

0 2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0 F

Time (hours)

Ven

om

Mass

(m

g)

0 2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0 E

Time (hours)

Ven

om

Mass

(m

g)

0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0 C

Time (hours)

Ve

no

m M

ass

(m

g)

0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0 D

Time (hours)

Ve

no

m M

ass

(m

g)

0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0 A

Time (hours)

Veno

m M

ass

(m

g)

0 2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0 B

Time (hours)

Ven

om M

ass

(m

g)