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Keywords:

  • Epilepsy;
  • Rodents;
  • Elimination rate;
  • Pharmacoresistance;
  • Epileptogenesis;
  • Dosing regimens

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

Summary:  Rodent models of chronic epilepsy with spontaneous recurrent seizures likely represent the closest parallel to the human condition. Such models may be best suited for therapy discovery for pharmacoresistant epilepsy and for antiepileptogenic or disease-modifying therapeutics. However, the use of such rodent models for therapy discovery creates problems with regard to maintaining effective drug levels throughout a prolonged testing period. This is particularly due to the fact that rodents such as rats and mice eliminate most drugs much more rapidly than humans. Thus, knowledge about elimination rate of a test drug in a laboratory species is essential for development of a treatment paradigm that allows maintaining adequate drug levels in the system over the period of treatment. Currently, the most popular models of epilepsy with spontaneous seizures are poststatus epilepticus models of temporal lobe epilepsy in rats. Such models are both used for studies on antiepileptogenesis and drug resistance. For validation of these models, current antiepileptic drugs (AEDs) have to be used. In this article, the elimination rates of these AEDs and their effective plasma levels in rats are reviewed as a guide for developing treatment protocols for chronic drug testing. The advantages and disadvantages of several technologies for drug delivery are discussed, and some examples for calculation of adequate treatment protocols are given. As shown in this review, because of the rapid elimination of most AEDs in rats, it is no trivial task to maintain effective steady-state AED levels in the plasma throughout the day over multiple days to ensure that there will be adequate levels in the system for the purpose of the experiment. However, the use of an adequate dosing regimen that is based on elimination rate is an absolute prerequisite when using rat models for discovery of new antiepileptogenic therapies or therapies for pharmacoresistant epilepsy, because otherwise such models may lead to erroneous conclusions about drug efficacy.

Antiepileptic drugs (AEDs) are the most important therapeutic means of preventing recurrence of seizures in patients with epilepsy. AEDs do not cure the condition but will often control seizures completely if the AEDs are taken regularly. For this purpose, AEDs are administered daily with dosage intervals depending on the elimination half-life of the drug in order to achieve and maintain effective steady-state drug concentrations throughout the day (Eadie, 1999). Most AEDs in clinical use have half-lives in humans of 8 to >24 h, so that antiepileptic concentrations can be maintained by one to three drug applications per day (Mattson, 2002). Monitoring of AED concentrations may be useful for management of drug therapy (Eadie, 1999).

Although significant advances have been made in the treatment of epilepsy over the past decades, about one third of patients with epilepsy remains resistant to current pharmacotherapies (Löscher and Potschka, 2005). Even in patients in whom pharmacotherapy is efficacious, current AEDs do not seem to affect the progression or underlying natural history of epilepsy (Löscher and Schmidt, 2002). Furthermore, there is currently no drug available which prevents the development of epilepsy, e.g., after head trauma (Temkin et al., 2001; Schachter, 2002). Thus, for improving therapy of epilepsy, there are at least three important goals for the future (Löscher and Schmidt, 2006a): (1) better understanding of processes leading to epilepsy, thus allowing to create therapies aimed at the prevention of epilepsy in patients at risk; (2) improved understanding of biological mechanisms of pharmacoresistance, allowing to develop drugs for reversal or prevention of resistance; and (3) development of disease-modifying therapies, inhibiting the progression of epilepsy. For achieving these goals, the existing process of therapy discovery and development should be improved with a focus on developing preclinical animal models of epileptogenesis and pharmacoresistance (Stables et al., 2003).

However, one major difficulty in this regard is that laboratory animal species such as mice or rats eliminate most AEDs and various other drugs much more rapidly than humans, which is often not considered when such drugs are used in experimental rodent studies (Löscher and Schmidt, 1988). When AEDs are used at single doses in animal experiments, as for instance when assessing the acute anticonvulsant potency of such drugs in seizure tests, rapid elimination is not a problem as long as the drugs are tested at their individual time of peak effect after oral or parenteral (e.g., i.p.) administration (Löscher and Schmidt, 1988). However, when drugs are administered over a prolonged period of time, knowledge of elimination rate is essential to achieve and maintain effective steady-state drug concentrations during such an experiment. As such, this is not different from treatment strategies in patients with epilepsy. However, whereas elimination half-lives of AEDs in humans are widely known and used for appropriate dosing protocols, this is not the case for elimination half-lives of AEDs in mice or rats.

EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

Maintaining effective AED levels throughout a prolonged testing period in mice or rats is important for preclinical therapy evaluation because of several reasons.

(1) With some drugs the anticonvulsant efficacy increases during prolonged treatment (Löscher and Schmidt, 1988); examples are primidone (due to accumulation of phenobarbital [PB]), valproic acid (VPA; reasons are unknown) and vigabatrin (VGB; due to accumulation of GABA by irreversible inhibition of its degradation). Consequently, determination of acute potency of such drugs underestimates their potency during prolonged treatment and, in case of new compounds, may thus lead to false decisions with respect to further preclinical or clinical development.

(2) With several AEDs, particularly benzodiazepines, the anticonvulsant efficacy decreases during prolonged treatment due to development of adaptive processes (“functional tolerance”) in the brain (Löscher and Schmidt, 2006b). With some older AEDs, such as PB, carbamazepine (CBZ) or phenytoin (PHT), “metabolic tolerance” may also occur due to enhanced drug elimination by induction of AED metabolizing enzymes. Tolerance is clinically advantageous when it concerns the adverse effects of AEDs but disadvantageous when it involves the antiepileptic efficacy itself. In mice and rats, tolerance to the anticonvulsant and adverse effect of benzodiazepines (BZDs) and various other AEDs can be demonstrated in a variety models of seizures or epilepsy with 1–4 weeks of daily drug administration, provided that effective drug concentrations are maintained during treatment (Löscher and Schmidt, 2006b).

(3) Prolonged drug administration may provide indications for the possible development of drug dependence when the animals are closely observed (body temperature, body weight, seizure threshold, anxiety-related behaviors etc.) for some days after drug withdrawal (File and Andrews, 1993; Bhargava, 1995). Such withdrawal symptoms have for instance been described after prolonged administration of BZDs, PB or VGB in rats (Löscher and Hönack, 1989; Gibson et al., 1990; File and Andrews, 1993).

(4) By use of models of acquired epilepsy such as kindling or poststatus epilepticus (post-SE) models of temporal lobe epilepsy (TLE), in which the progressive process leading to epileptogenesis can be studied, prolonged treatment with drugs during this process may identify drugs that provide antiepileptogenic or disease-modifying effects (Stables et al., 2003). For instance, we have recently reported that prolonged administration of VPA after SE prevents the development of hippocampal damage, including the loss of dentate hilar neurons, and most of the behavioral alterations associated with epileptogenesis (Brandt et al., 2006).

(5) Finally, for developing and validating new models of pharmacoresistant epilepsy, the maintenance of effective drug levels throughout a prolonged testing period is an absolute prerequisite in order to avoid that animals are falsely considered pharmacoresistant. Based on the recommendations of a models workshop organized by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) in 2002 (Stables et al., 2003), pharmacoresistance in animal models can be minimally defined as persistent seizure activity not responding or with very poor response to at least two current AEDs at maximum tolerated doses.

For discovery of new therapeutics for pharmacoresistant epilepsy and for disease-modification of epilepsy, rat models of injury-induced epilepsy based on electrically or chemically induced SE have been recommended by the participants of the recent NIH models workshop (Stables et al., 2003). New rat models of pharmacoresistant epilepsy have to be validated by prolonged treatment with available AEDs, so that knowledge of their elimination kinetics in rats is important. The purpose of this review is to summarize the differences in elimination kinetics of AEDs in humans and rats as a guide for treatment protocols in post-SE models of TLE. Furthermore, effective plasma AED levels in rats are described, and some examples for calculation of adequate treatment protocols are given.

ELIMINATION HALF-LIVES OF AEDS IN RATS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

Table 1 summarizes elimination half-lives of 18 AEDs and/or their major active metabolites in adult rats. For comparison, half-lives of these drugs in humans are shown. Data were taken from the literature, including various studies of the author's own laboratory, in which elimination half-life was determined in groups of rats after i.p. administration of a single dose. Eleven of the 18 AEDs have half-lives of >8 h in humans, whereas this is the case for only 3 AEDs in rats (P = 0.0153 by Fisher's exact test). The minimum half-life of all AEDs or the major active metabolites in humans is at least 5 h, while this criterium is only met by 5 AEDs in rats (P = 0.0455). Indeed, without exception, all AEDs are more rapidly eliminated by rats than by humans. For AEDs with half-lives below 5 h in rats (12 of the AEDs shown in Table 1), it is almost impossible to maintain effective drug levels by intermittent (e.g., three times daily) daily drug administration, except the drug exhibits nonlinear (saturation) kinetics, so that the half-life increases upon repeated administration. The most important example in this respect is PHT, which will be discussed later. As a consequence, based on the half-lives shown in Table 1, only 5 AEDs (ethosuximide, lamotrigine, PB, PHT, zonisamide) have half-lives allowing maintenance of effective drug levels during prolonged treatment by conventional routes of administration, i.e., two to three times daily i.p. or oral administration. Furthermore, maintenance of anticonvulsant efficacy during prolonged treatment is possible with VGB, because its duration of anticonvulsant action is independent of elimination half-life, due to irreversible inhibition of GABA degradation (Connelly, 1993). With all other AEDs shown in Table 1, conventional routes of treatment, even with three times daily administration, will not allow maintenance of effective drug concentrations in rats. This is similar for mice, although less pharmacokinetic data are available for this species (e.g., van der Klejin et al., 1971; Löscher and Esenwein, 1978; Leal et al., 1979; Caccia et al., 1980; Bourgeois et al., 1982; Iven and Feldbusch, 1983; Atlas et al., 1980; Radulovic et al., 1995; Benedetti et al., 2004; Caldwell et al., 2005). Mice often eliminate AEDs even more rapidly than rats. For instance, elimination half-lives are 7.5 h for PB, 1 h for ethosuximide, and 0.8 for VPA, respectively. Thus, all recommendations of this review for developing treatment protocols for rats equally apply for mice.

Table 1. A comparison of elimination half-lives of antiepileptic drugs in humans and rats. Data for humans are from Levy et al. (2002) and Neels et al. (2004). References for rat data are given in the table.
AEDHalf-life (h)References for the rat data
HumanRat
  1. aActive metabolites; bshortens on continuing exposure to the drug (because of enzyme induction); cnonlinear kinetics (half-life increases with dose); dduration of action independent of half-life because of irreversible inhibition of GABA degradation. Abbreviations: DMD, desmethyldiazepam; MHD = monohydroxy derivative

Carbamazepine25—50a, b1.2–3.5aFarghali-Hassan et al., 1976; Hönack and Löscher, 1989
Clobazam16—501Löscher and Rundfeldt, 1990
Diazepam24—72a(DMD = 40-130)1.4a(DMD = 1.1)Löscher and Schwark, 1985
Ethosuximide40–6010–16Teschendorf and Kretschmar, 1985
Felbamate14-222-17cAdusumalli et al., 1991
Gabapentin5-72-3Bartoszyk et al., 1986
Lamotrigine21-5012->30Miller et al., 1986; Walker et al., 2000
Levetiracetam6-112-3Löscher and Hönack, 1998; Doheny et al., 1999
Oxcarbazepine1-2.5a?aGram and Philbert, 1986
(MHD = 8-14)(MHD = 0.7-4) 
Phenobarbital70-1009-20Hoffmann and Levy, 1989; Löscher and Hönack, 1989;
Brandt et al., 2004
Phenytoin15-20c∼1-8cGerber et al., 1971; Rundfeldt and Löscher, 1993
Pregabalin62.5Welty et al., 1997
Primidone6-12a5aLöscher and Hönack, 1989
Tiagabin5-81Wang et al., 2004
Topiramate20-302-5R.P. Shank, unpublished data
Valproate8-15a∼1-5a, cLöscher, 1978; Dickinson et al., 1979
Vigabatrin5-7d∼1dRundfeldt and Löscher, 1992
Zonisamide60-708Taylor et al., 1986

EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

Most data shown in Table 1 are from either Sprague–Dawley or Wistar rats, i.e., the most widely used outbred rat strains in biomedical research. Outbred and inbred rat strains may differ in pharmacokinetics because of genetic differences in drug metabolism (Kacew and Festing, 1996). Furthermore, differences in pharmacokinetics may exist in different outbred rat strains or even within the same strain obtained from different breeders. This has not been systematically investigated for AEDs, so that the half-lives shown in Table 1 may differ between rat strains. Only a few AEDs have been compared in Wistar and Sprague–Dawley rats. One example in this regard is PB. In a group of female Wistar rats, an average half-life of 12.4 h was determined after i.p. injection of 30 mg/kg PB (Löscher and Hönack, 1989). When the same dose was administered in female Sprague–Dawley rats, an average half-life of 14.1 h was determined (Fig. 1), which is in the same range as the half-life determined in Wistar rats. Similarly, the elimination kinetics of phenytoin were comparable in the two rat strains (Rundfeldt and Löscher, 1993; Bethmann et al., 2007).

image

Figure 1. Pharmacokinetics of phenobarbital (PB) in rats. (A) PB was i.p. administered in female Sprague–Dawley rats at three dose levels, i.e., 20, 25, and 30 mg/kg, using two rats per dose. Average plasma levels determined at several times after administration of these doses are shown. The lower border of the therapeutic plasma concentration range of PB in epilepsy patients (10–40 μg/ml) is indicated by the hyphenated line. Average elimination half-lives determined from the plasma concentration versus time curves were 20.4 h (20 mg/kg), 16.2 h (25 mg/kg), and 14.1 h (30 mg/kg), respectively. Based on the plasma levels and average elimination rate of PB determined in this experiment, a dosing regimen for prolonged treatment was calculated with the aim to maintain PB concentrations in plasma in the range of 20–40 μg/ml over 24 h per day. (B) The protocol obtained from this calculation (using the simulation methods of WinNonlin; see text) was tested in 6 rats over one week, resulting in plasma levels (shown as mean ± SEM) within or above the desired range when blood was sampled immediately before and 1 h after the last injection in the morning of day 8. (C) Subsequently, this protocol was used for prolonged treatment of 11 epileptic rats with spontaneous recurrent seizures. These rats were treated over two weeks, again resulting in plasma concentrations (shown as mean ± SEM) within the desired range. Data are from Brandt et al. (2004).

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Apart from strains, gender may affect elimination half-lives of various drugs in rats, which is due to sex-related differences in cytochrome P-450 enzymes in liver microsomes (Kato and Kamataki, 1982; Skett, 1988; Kato and Yamazoe, 1992). Several drugs are eliminated much slower by female rats compared to male rats, but again this has not been systematically investigated for AEDs. Among the most widely studied drugs in terms of gender differences in rodents are the barbiturates. As early as 1937, it was observed that female rats slept longer than males after administration of certain barbiturates (Holck et al., 1937). It is now held that this difference is a consequence of gender differences in the elimination kinetics of these drugs (Kato and Kamataki, 1982; Skett, 1988; Kato and Yamazoe, 1992). For instance, in Lewis rats, the average elimination half-life of heptabarbital is 93 min in females but only 9.6 min in males, a striking and highly significant difference (Hoffman and Levy, 1989). A less marked gender difference was determined for PB with a half-life of about 8.5 in male and 11 h in female Wistar rats (Hoffman and Levy, 1989).

Farghali-Hassan et al. (1976) studied the pharmacokinetics of CBZ in male and female Sprague–Dawley rats. Elimination half-life after a single dose of 25 mg/kg i.v. was 111 (90–132) min in females, but only 70 (62–78) min in males. The authors suggested a difference in drug degradation at the microsomal level, because it is known that, in rats, the rate of various drugs by liver microsomal enzymes is higher in males than in females (Farghali-Hassan et al., 1976).

A two-fold gender difference in elimination half-life was found for topiramate (TPM) in Sprague–Dawley rats (R.S. Shank, unpublished data). After i.v. administration of 15 mg/kg, average half-life was 2.2 h in male rats, but 5.2 h in female rats.

In a large study in 20 male and 27 female amygdala-kindled Wistar rats, in which PHT was administered at a dose of 75 mg/kg i.p., we determined its effects on afterdischarge threshold (ADT) in a total of 78 trials in males and 104 trials in females (Ebert et al., 1994). Blood was sampled immediately after ADT determination, i.e., 60 min after drug administration, for PHT analysis in plasma. PHT proved to be much more efficacious to increase ADT in females than in males. Furthermore, female rats had significantly higher plasma levels than males (27.4 ± 0.67 vs. 21.2 ± 0.48; P < 0.001), demonstrating that female rats eliminate PHT more slowly than males (Ebert et al., 1994). Thus, gender may affect both antiepileptic efficacy and elimination kinetics of AEDs in rats. Female rats have clear advantages for prolonged administration of AEDs, because most drugs that are eliminated by liver enzymes have higher half-lives in females than in males, thus favoring maintenance of effective drug levels during prolonged drug administration.

EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

When developing a treatment protocol for maintenance of effective plasma levels of AEDs in rats, it is of course necessary to know which plasma levels of AEDs are associated with anticonvulsant efficacy in this species. Table 2 illustrates the “therapeutic plasma concentration range” of various AEDs in patients with epilepsy and also gives the effective concentration range in rats determined in rat models of seizures or epilepsy. As can be seen from this table, effective plasma AED levels are remarkably similar in humans and rats. However, because of the marked differences in elimination kinetics of AEDs between humans and rats (Table 1), much higher doses of AEDs have to be administered to achieve and maintain such effective AED levels in rats than in humans. Furthermore, because of the rapid elimination, most AEDs have to be administered either more frequently over the day than in man or have to be given continuously to allow maintenance of effective levels during prolonged treatment.

Table 2. Effective (“therapeutic”) ranges of plasma concentrations for established AEDs in patients with epilepsy. For comparison, AED concentrations determined in plasma either at anticonvulsant doses (ED50s) in the maximal electroshock seizure (MES) or pentylenetetrazole (PTZ) tests or during prolonged treatment of epileptic rats are shown. Data for humans are from Eadie (1999), Levy et al. (2002), and Neels et al. (2004). References for rat data are given in the table.
AEDRange of effective (“therapeutic”) concentrations (μg/ml)References
Epilepsy patientsRats
After single dose administration in the MES or PTZ tests (EC50)After prolonged administration in rats with SRS or induced seizures
  1. DMD, desmethyldiazepam; EC50, effective plasma concentration determined after application of ED50 at time of seizure test; MHD, monohydroxy derivative; SRS, spontaneous recurrent seizures.

Carbamazepine4–124–6 (MES)16–22 (kindled seizures)Hönack and Löscher, 1989; Löscher et al., 1991a
Clobazam0.1–1.4 (desmethylclobazam = 0.3–0.5) 0.5–0.7 (kindled seizures)Löscher and Rundfeldt, 1990
Diazepam0.3–0.5 (DMD = 0.3–0.5)MES: 0.2–2.3 (DMD = 0.12–0.6); PTZ: 0.08–0.120.2–0.3 (kindled seizures)(DMD =∼0.1)Löscher and Schwark, 1985; Löscher et al., 1991a, 1991b
Ethosuximide40–100108–134 (PTZ) Löscher et al., 1991b
Felbamate30–80 
Gabapentin12–20 
Lamotrigine3–143–5 (MES)3–15 (SRS)Castel-Branco et al., 2003; C. Brandt and W. Löscher, unpublished data
Levetiracetam10–37 33–61 (SRS)Glien et al., 2002
Oxcarbazepine3–40 (MHD) 
Phenobarbital10–4013–29 (MES)14–27 (SRS)Löscher et al., 1991a; Brandt et al., 2004
Phenytoin10–207–12 (MES)6–23 (SRS)Löscher et al., 1991a; van Vliet et al., 2006; Bethmann et al., 2007
Pregabalin3–8 
Primidone5–12 (plus 15–40 phenobarbital) 20–33 (kindled seizures)(phenobarbital 7–50)Löscher and Hönack, 1989
Tiagabin10–100 
Topiramate5–25 
Valproate50–100240–270 (MES); 210–298 (PTZ)50–100 (PTZ)Löscher et al., 1991a, 1991b; Löscher and Hönack, 1995
Vigabatrin1–36 <1 (kindled seizures)Rundfeldt and Löscher, 1992
Zonisamide10–38 

INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

One of the initial issues in a laboratory drug study is the choice of an adequate drug vehicle and formulation for administering the drug, since both administration vehicles and drug formulations can markedly affect the bioavailability of drugs (Löscher et al., 1990; Castel-Branco et al., 2002). In general, AEDs have been made lipophilic to enable them to cross the blood–brain barrier and to reach their brain targets. Consequently, most AEDs have a poor water solubility, unless they are available in the form of water-soluble salts, such as sodium VPA or sodium PB, or can be transferred to a water-soluble salt, such as PHT and diazepam (by means of dilute HCl). AEDs with poor water solubility either have to be administered in the form of aqueous suspensions or can be dissolved in lipophilic vehicles, such as polyethylene glycol 400 (PEG 400) or glycofurol, and then diluted by water (Löscher et al., 1990). When injected i.p., drug suspensions are usually associated with low and variable bioavailability and a retarded time of peak drug effect compared to drug solutions (Löscher et al., 1990; Castel-Branco et al., 2002). Thus, drug solutions are generally preferable to drug suspensions. When lipophilic vehicles are used for dissolving a drug in water, the vehicle can exert effects of its own and change the efficacy of the dissolved drug (Löscher et al., 1990). An exception is PEG 400 in concentrations up to 30% (in water), which does not seem to exert any effects on seizure thresholds or behavior of rodents (Löscher et al., 1990). An alternative to lipophilic vehicles for dissolving a drug are amorphous cyclodextrin derivatives, which form a water-soluble inclusion complex with the drug (Pitha et al., 1988; Löscher et al., 1995). For instance, cyclodextrins have been used to form a water-soluble formulation of CBZ, which was much better tolerated than a glycofurol-based formulation (Löscher et al., 1995). Irrespective of the administration vehicle or drug formulation used in a study with administration of an AED in a rodent model of epilepsy, the drug's bioavailability should be controlled by determining the concentration of the AED in plasma or brain to avoid misinterpretations because of poor drug absorption. Even if the drug is administered as aqueous solution, both bioavailability and time of peak drug effect may be dose dependent, with retarded absorption after administration of high doses (Löscher et al., 1990). Furthermore, if the stability of the drug formulation used for an animal experiment is not known, the formulation should be prepared freshly before each drug application.

CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

As discussed above, when using conventional routes of administration, such as three times daily i.p. or oral drug administration, a half-life of at least 5 h is considered necessary for allowing maintenance of effective drug levels during prolonged AED administration in rats. If the half-life of an AED in rats is known, how can treatment protocols for prolonged drug administration be calculated, so that effective drug levels are reached and maintained? An example is shown in Fig. 1. In this experiment, our goal was to develop a treatment protocol for PB allowing maintenance of plasma drug levels of 20–40 μg/ml in rats, i.e., plasma levels within or above the “therapeutic range” (10–30 μg/ml) in patients with epilepsy (Eadie, 1999). In a first step, the elimination half-life of PB was determined after single dose administration in female Sprague–Dawley rats, i.e., the rat strain and gender which we intended to use for the chronic experiment. As shown in Fig. 1A, three different doses of PB (20, 25, and 30 mg/kg i.p.) were administered in groups of two rats, resulting in half-lives of 13–20 h in the 6 rats (mean 16.9 ± 1.4 h). Next, WinNonlin® (Pharsight Corp., Mountain View, CA, U.S.A.), a PC-based pharmacokinetic/pharmacodynamic data analysis program (Gabrielsson and Weiner, 1999), was used for nonlinear curve fitting of the time-concentration data shown in Fig. 1A. This program also allows a simulation to generate multiple-dose concentrations based on parameters from fitting single-dose data. By simulation of a compiled library model, we modeled the bolus and maintenance doses to achieving and maintaining PB plasma levels in the range of 20–40 μg/ml. Based on this pharmacokinetic modeling by WinNonlin, the following dosing regimen was proposed by the program: a bolus dose of 25 mg/kg i.p., followed 10 h later by 15 mg/kg i.p., and then twice-daily 15 mg/kg i.p. (at 8 a.m. and 6 p.m.) on subsequent weeks (Brandt et al., 2004). This dosing regimen was used for a treatment period of one week in six rats. As shown in Fig. 1B, after one week of treatment, trough levels of PB before the final drug administration were within the limits (20–40 μg/ml) on which our calculations were based. Therefore, the treatment protocol was used in 11 rats with spontaneous recurrent seizures (SRS), which were treated over two weeks, again resulting in plasma levels of PB with the therapeutic range (Fig. 1C). This example illustrates that pharmacokinetic modeling allows adequate calculations of treatment protocols for maintenance of effective drug levels during prolonged treatment, provided the half-life of the drug to be evaluated is known.

However, such pharmacokinetic modeling is much more complicated if nonlinear kinetics are involved, because the extent of saturation of metabolizing enzymes upon repeated drug administration is difficult to model. I will use PHT to illustrate the problem of establishing a dosing regimen in rats for AEDs with dose-dependent elimination kinetics. Phenytoin is eliminated almost entirely by metabolic transformation by cytochrome P450 (CYP) isoforms CYP2C9 and CYP2C19 (Browne and LeDuc, 2002). Metabolism by these enzymes exhibits nonlinear enzyme kinetics, so that the elimination rate of PHT is dose dependent (Browne and LeDuc, 2002). For instance, after i.v. injection of PHT in male Sprague–Dawley rats, half-lives of 0.6, 1.2 and 2.5 were calculated at doses of 10, 25 and 40 mg/kg, respectively (Gerber et al., 1971). In addition to the saturation of metabolism by high levels of PHT, the major phenytoin metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), inhibits the hydroxylation of PHT in rats (Ashley and Levy, 1972). As a consequence, upon repeated administration of PHT in rats, the time to reach steady-state plasma concentrations varies nonlinearly with the dose level and dosing rate. We have demonstrated in female Wistar rats that, during repeated administration of PHT, inhibition of metabolizing enzymes is long-lasting, leading to reduced elimination of subsequent doses (Rundfeldt and Löscher, 1993). As a consequence of the resulting drug accumulation and neurotoxicity, development of treatment protocols for prolonged treatment with PHT in rats proved to be much more complicated than with other AEDs. One advantage of the saturation kinetics is that effective plasma drug levels could be maintained by once-daily treatment. However, during prolonged treatment, enzyme induction by PHT develops in rats, which counteracts at least in part the advantage of saturation kinetics for maintaining effective drug levels (Rundfeldt and Löscher, 1993). In Fig. 2 we illustrate some of our experiments in female Wistar rats, which we performed to develop a dosing regimen allowing maintenance of PHT levels in plasma within or above the therapeutic range (10–20 μg/ml; Eadie, 1999) of this AED in kindled rats. As shown in Fig. 2A, PHT was eliminated more rapidly after single dose administration of 50 mg/kg (t0.5∼1.5 h) than 75 mg/kg (t0.5∼4 h), which can be explained by the decrease in drug clearance upon increase in dosage. However, when 50 mg/kg was injected one day after administration of 75 mg/kg, elimination was much slower compared to administration of 50 mg/kg alone, so that plasma levels within the therapeutic range were maintained for about 8 h (Fig. 2A). Treatment of rats over two weeks with this dosing regimen (bolus dose 75 mg/kg, followed by once daily administration of 50 mg/kg) resulted in therapeutic plasma levels at either 1 or 6 h after each dosing over the whole period of treatment, although plasma levels declined by the end of this period because of metabolic tolerance (Fig. 2B). This dosing regimen was recently used by van Vliet et al. (2006) in a post-SE model of TLE in male Sprague–Dawley rats. As in our experiments in Wistar rats (Rundfeldt and Löscher, 1993), therapeutic plasma levels of PHT (10–20 μg/ml) were obtained for about 8 h after each dosing with this protocol (van Vliet et al., 2006). Therefore, van Vliet et al. (2006) recorded the effect of PHT on spontaneous seizures only during this time window (from 9 a.m. to 5 p.m.) each day over a period of one week, resulting in a significant decrease in seizure frequency compared to predrug control recordings.

image

Figure 2. Pharmacokinetics of phenytoin (PHT) in rats. In all graphs, the lower border of the therapeutic plasma concentration range of PHT in epilepsy patients (10–20 μg/ml) is indicated by the hyphenated line. (A) PHT was i.p. administered in female Wistar rats at doses of 50 mg/kg (6 rats), 75 mg/kg (5 rats), and 50 mg/kg 24 h after injection of 75 mg/kg (7 rats). Average plasma levels after these doses are shown. Following administration in PHT-naive rats, PHT was eliminated much more rapidly after 50 than 75 mg/kg, indicating saturation of drug metabolism by increasing dosage. However, when 50 mg/kg were injected 24 h after 75 mg/kg, the elimination rate was almost indistinguishable from that of 75 mg/kg, indicating long-lasting saturation of drug metabolism after the first drug administration. (B) Based on these observations, we injected PHT once daily (75 mg/kg at day 1 and 50 mg/kg at all subsequent days) over two weeks in nine fully kindled rats. Effective drug levels in the range of 10–27 μg/ml (shown as means ± SEM) were maintained for 6 h each day over the duration of prolonged treatment. However, at the end of the second week, plasma levels were significantly lower (P < 0.01) compared to the other days, indicating induction of metabolism. The PHT levels shown were associated with significant anticonvulsant activity throughout the period of treatment. Data are from Rundfeldt and Löscher (1993). (C) In order to prolong the maintenance of effective PHT levels to 24 h per day, we subsequently modified the protocol in Sprague–Dawley rats by treating the rats twice daily with PHT (50 mg/kg in the morning and 25 mg/kg in the evening) following the bolus dose of 75 mg/kg on the first day. (Bethmann et al., 2007). This protocol was tested in two rats over one week (unpublished data: K. Bethmann, C. Brandt, W. Löscher), resulting in average plasma levels within or above the desired range, when plasma concentrations of PHT were determined either 1 h before or 1 h after the administration in the morning or evening. Please note that the “1 h before” data are 11 h after the last injection of PHT, because the dosing interval was 12 h in this experiment. Subsequently, this protocol was used for prolonged treatment of epileptic rats with spontaneous recurrent seizures (Bethmann et al., 2007).

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In addition to the dosing regimen illustrated in Fig. 2B, which was used by us in kindled rats (Rundfeldt et al., 1993) and van (Vliet et al., 2006) in epileptic rats, we tested several other dosing protocols in Wistar rats (Table 3). Most of these protocols resulted in too high plasma levels of PHT, associated with severe toxicity. Toxicity typically developed with 2–3 days of treatment because of marked retardation of PHT elimination. For instance, when PHT was injected once daily at 75 mg/kg, half-lives after the second and third dosings were about 20 h compared to 4 h after the first dosing (Rundfeldt and Löscher, 1993). More recently, we tested various dosing regimens of PHT in female Sprague–Dawley rats (Bethmann et al., 2007). Results were similar to those previously obtained in female Wistar rats (Table 3). The most promising dosing regimen for maintenance of effective plasma levels of PHT was starting treatment by a bolus dose of 75 mg/kg, followed from the second day on by 50 mg/kg in the morning and 25 mg/kg in the evening (Table 3; Fig. 2C). This regimen provided plasma levels within the therapeutic range over 24 h per day. However, in contrast to naive rats, in epileptic rats this protocol produced toxicity, including proconvulsant effects, so that doses had to be individually adapted in each rat (Bethmann et al., 2007). A decreased tolerability of epileptic rats was also observed for LTG (C. Brandt and W. Löscher, unpublished observations), so that transferring dosing regimens from normal to epileptic rats may necessitate dose-adjustment for some AEDs. In this respect it is interesting to note that decreased tolerability of AEDs was not only observed in epileptic rats but also in fully kindled rats, indicating that the chronic brain alterations associated with epileptogenesis render the brain more sensitive to adverse effects of certain drugs (Hönack and Löscher, 1995).

Table 3. Experiments with phenytoin in female Wistar or Sprague–Dawley rats aimed to establish a dosing protocol that allows maintenance of effective plasma levels (10-20 μg/ml) during prolonged treatment
Rat strainExperiment #Dosing protocol (all doses in mg/kg)Maintenanceof effective plasma levels (μg/ml)Toxicity (necessitating termination of experiment)
  1. At least two rats were used per protocol. Toxicity necessitating termination of the experiment was characterized by loss of righting reflex with rats laying on their sides, severe hypothermia, opisthotonus, tonic extension of hind legs, seizures and, eventually, death. Data are from Rundfeldt and Löscher (1993) and Bethmann et al. (2007).

Wistar13 times daily 75 i.p.Too high levels (>40)Yes
Wistar23 times daily 50 i.p.Too high levels (> 40)Yes
Wistar375 i.p. on first day, then 3 times daily 50 i.p.Too high levels (> 40)Yes
Wistar4Once daily 75 i.p.Too high levels (> 40)Yes
Wistar575 i.p. on first day, then once daily 50 i.p.Yes (but only for about 8 h after each dosing)No
Sprague–Dawley175 i.p. on first day, then once daily 50 i.p.Yes (but only for about 8 h after each dosing)No
Sprague–Dawley275 s.c. on first day, then once daily 50 s.c.Yes (but only for about 8 h after each dosing)No
Sprague–Dawley375 i.p. on first day, then 2 times daily 50 i.p.Too high levels (> 40)Yes
Sprague–Dawley4Twice daily 50 i.p.Too high levels (> 40)Yes
Sprague–Dawley575 i.p. on first day, then daily 50 i.p. in the morning and 25 i.p. in the evening.YesNo
Sprague–Dawley675 i.p. on first day, then daily 40 i.p. in the morning and 25 i.p. in the evening.YesNo
Sprague–Dawley775 i.p. on first day, then twice daily 35 i.p.YesNo

For AEDs, such as VPA or CBZ, with short half-lives in rats (Table 1), one may try to achieve effective plasma levels during prolonged treatment by three times daily injection of high doses, provided this is not associated with too high toxicity. For instance, Leite and Cavalheiro (1995) administered CBZ three times daily (at 8:00, 11:00, and 2:00) at 40 mg/kg i.p. in male Wistar rats, resulting in a significant suppression of spontaneous seizures in the pilocarpine model of TLE. Similarly, three times daily administration of VPA at 200 mg/kg i.p. suppressed spontaneous seizures, whereas three times daily 150 mg/kg were not effective in this regard (Leite and Cavalheiro, 1995). However, the authors did not use continuous, 24-h EEG/video recordings in their study, but recorded spontaneous seizures only between 8 a.m. to 6 p.m., i.e., the time period that they covered by their AED administrations at 8 a.m., 11 a.m., and 2 p.m. In other words, it is not possible to maintain effective AED levels with such treatment protocols over 24 h, even when such high doses are used. However, as shown by the experiments of van Vliet et al. (2006); Leite and Cavalheiro (1995), recording of SRS for only a limited time each day, i.e., the time window in which effective drug levels are present, is an alternative to match effective AED levels and SRS for drugs with too short half-lives to allow maintenance of effective drug levels over 24 h.

In models with high frequency of spontaneous seizures, a possible alternative to prolonged drug administration is to test AEDs with short half-lives such as TPM (Table 1) only for a limited time (e.g., 6 h) after single administration of high doses (Grabenstatter et al., 2005). Using a crossover protocol (in which each rat served as its own control), Grabenstatter et al. (2005) examined the efficacy of TPM on spontaneous epileptic seizures in the kainate model of TLE in Sprague–Dawley rats. TPM dose-dependence reduced seizure frequency at doses of 10, 30, and 100 mg/kg compared to saline control sessions. More recently, Grabenstatter et al. (2006) used this approach also for CBZ. However, one of the problems associated with this approach is that spontaneous seizures in rats do not occur with a homogeneous distribution over 24 h or 7 days per week, but, as in patients with epilepsy, show large intra- and interanimal variation in their temporal appearance and may occur in clusters. Thus, for truly assessing the antiepileptic efficacy of a drug on SRS, there is no alternative to prolonged drug administration. When one wishes to maintain effective plasma levels of drugs with short half-lives in rats throughout 24 h per day over a period of prolonged treatment, i.e., conditions comparable to treatment of patients with epilepsy, routes for continuous AED administration have to be used.

ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

It is often argued that administration of drugs via the drinking water allows maintenance of effective drug levels in mice or rats. Indeed, for water-soluble AEDs with half-lives of at least 5 h, administration via the drinking water may be a very convenient way of prolonged drug application, provided that the palatability of the drinking water is not too much affected by the drug, resulting in reduced drinking by the animals (Löscher and Schmidt, 1988). Drugs that are not water soluble may be administered via the food. However, for drugs with shorter elimination half-lives, administration via the drinking water (or food) is not advantageous versus intermittent drug application, because rodents drink (or eat) mostly during the night, so that drug levels during day time will become too low for adequately assessing chronic drug efficacy (Löscher and Schmidt, 1988). As recently reported by Ali et al. (2006) for CBZ, this problem may be overcome by administering extremely high doses of an AED (e.g., 300 mg/kg/day CBZ) via the food, resulting in suppression of spontaneous seizures in the kainate model of TLE. However, Ali et al. (2006) did not report quantification of adverse effects or drug plasma levels associated with such treatment paradigm.

For most drugs that are rapidly eliminated, special administration techniques have to be used for maintaining effective levels, such as constant rate application via subcutaneously implanted osmotic minipumps or subcutaneous injection of special drug depot preparations. Osmotic pumps (such as the Alzet pumps produced by Durect Co., Cupertino, CA, U.S.A.) are miniature, implantable pumps used for research in mice, rats, and other laboratory animals (Perkins et al., 1999; Urquhart, 2000). These infusion pumps continuously deliver drugs, hormones, and other test agents at controlled rates from one day to several weeks without the need for external connections or frequent handling. Their unattended operation eliminates the need for repeated nighttime or weekend dosing. The subcutaneous implantation of drug-containing osmotic minipumps is widely used for systemic drug administration and provides a convenient method for the chronic dosing of laboratory animals (Perkins et al., 1999). However, there are a number of problems that restrict the use of minipumps for continuous administration of AEDs. Ideally, the drug should be water soluble and stable in solution for the duration of the experiment. However, several AEDs are either not water soluble or stable in aqueous solution. For instance, BZDs such as diazepam can be dissolved in water by means of dilute HCl, but this aqueous solution has a too low stability for use in osmotic minipumps over an extended period of time (Löscher, 1986). In case of AEDs that are not water soluble, ALZET pumps are capable of delivering drugs that have been dissolved by means of vehicles such as PEG 300, PEG 400, propylene glycol, and glycerol, provided the solution is compatible with the interior reservoir of the pump. Also cyclodextrins may be helpful to increase solubility of drugs for continuous administration. As for water-soluble drugs, the stability of the AED solution for the duration of the planned experiment has to be proven. However, as discussed above, the use of lipophilic vehicles may affect the pharmacology of the drug to be tested. The use of drug suspensions in minipumps cannot be recommended, because suspensions usually precipitate in the pumps during the duration of delivery.

Another problem is the limited volume of the minipumps. For instance, an Alzet pump (2ML1) for rats, which provides continuous drug release with a pumping rate of 10 μl/h over one week, has a drug reservoir volume of 2.0 ml. Thus, the solubility of an AED, its anticonvulsant potency, and its half-life will determine whether effective drug levels can be obtained and maintained in rats under these conditions. An example is levetiracetam (LEV), which we previously administered by this type of osmotic minipumps in rats (Glien et al., 2002). Because of the extremely high water-solubility of the compound, 1000 mg could be dissolved in 2 ml of distilled water and filled in the minipumps, resulting in daily doses of about 400 mg/kg. As a result of the rapid elimination of LEV in rats (half-life 2–3 h; Table 1), this high daily dose resulted in average plasma concentrations of only about 40 μg/ml, which, however, is around the maximal plasma levels of LEV determined during twice-daily treatment of epilepsy patients with 1,000 mg of this drug (Patsalos, 2002), and was associated with antiepileptic efficacy in a post-SE model of TLE in rats (Glien et al., 2002). For treatment periods exceeding one week, the minipumps were replaced once per week by new minipumps filled with LEV (Glien et al., 2002). Too frequent replacement of pumps, however, may lead to irritation and inflammation of the subcutaneous tissue in the neck region, where the pumps are usually implanted in rats.

VPA is another example, in which osmotic minipumps are extremely useful for prolonged treatment of an AED which has a too short half-life for conventional treatment protocols with intermittent drug application. When used as sodium salt, VPA is highly water soluble. After single dose administration in rodents, effective plasma levels of VPA are >200 μg/ml in rats (Table 2). However, during prolonged administration of VPA, effective plasma levels are considerably lower (Table 2), most likely because of an increase of VPA's anticonvulsant activity during chronic administration that occurs in both humans and laboratory animals (Löscher, 2002). Thus, based on half-life (Table 1) and effective concentrations of VPA in rats (Table 2), it should be possible to maintain effective plasma concentrations via minipumps by daily doses of 400 mg/kg or above. For instance, prolonged treatment of rats with VPA via s.c. implanted osmotic minipumps (daily dose 875 mg/kg for seven days) has been used to study the effect of VPA on corticotropin-releasing factor systems in rat brain (Stout et al., 2001). In smaller rodent species, such as mice or gerbils, osmotic minipumps have also been successfully applied to maintain effective AED levels during prolonged treatment (Nau and Zierer, 1982; Nau, 1985; Löscher, 1986). For instance, during prolonged administration of VPA in epileptic gerbils, using Alzet 2ML2 pumps that deliver the drug over two weeks, daily doses of 500 mg/kg could be achieved, resulting in stable plasma concentrations of VPA and an increasing anticonvulsant effect of VPA over time (Löscher, 1986). Apart from LEV and VPA, s.c. implanted osmotic minipumps have been used to continuously administer the AED VGB after SE in order to study whether VGB prevents neuronal damage and epileptogenesis in rats (Pitkänen et al., 1999; Halonen et al., 2001).

Nau's group has developed an alternative device for continuous administration of VPA in rodents to overcome the dose limitation posed by the small volume of osmotic minipumps (Nau et al., 1983). They drilled various numbers of holes (3 mm diameter) into the conical portion of 0.5 ml disposable Eppendorf-microtubes, silastic tubing was pulled over these holes, VPA (the organic liquid, not the sodium salt) was then filled into the microtubes, which were closed with their stoppers. The number of 3 mm-holes and the thickness of the silastic tubing determined the VPA dose released. After s.c. implantion in mice, the devices resulted in persistent drug concentrations over several weeks (Nau et al., 1983). The drug reservoirs were easily refillable in situ, which greatly extended the duration of the experiments. Nau et al. (1983) proposed that the implantable and refillable drug reservoirs may be an appropriate device for pharmacological and toxicological studies of compounds with short half-lives and high clearance rate in rodents.

Another strategy for maintaining effective AED levels during prolonged treatment in rats is continuous constant-rate i.v. infusion through a chronically implanted i.v. catheter, which is connected to an infusion pump via a flexible tubing and swivel in freely moving animals. For instance, when VPA was i.v. infused at constant rate (600 mg/kg/day) in a volume of 24 ml/kg/day over several days, effective plasma levels and significant anticonvulsant effects were maintained in rats throughout the period of treatment (Löscher and Hönack, 1995). However, to achieve continuous i.v. administration in rats over several days or weeks is not trivial, but associated with a number of potential problems, such as clotting of the chronically implanted i.v. catheters. Clotting can be prevented by regularly flushing the catheter with heparinized solutions and the use of specifically coated tubings. Bertram et al. (2005) have used continuous i.p. infusion through a chronically implanted i.p. catheter in rats to test the efficacy of PB and PHT on SRS over two weeks. The drugs demonstrated efficacy with blood levels that were comparable to established human therapeutic ranges. However, the prolonged i.p. infusion of AEDs was associated with peritoneal irritation or even peritonitis resulting in death of several rats, so that this cannot be considered a useful approach for maintenance of steady-state AED levels in rats.

Infusions through an indwelling (chronically implanted) gastric tube have been successfully used in freely moving rats for prolonged drug administration. The advantage versus continuous i.v. infusion is that intragastric infusion is not limited to water-soluble drugs, but even drug suspensions can be administered in this way. Continuous intragastric infusion has been widely used for ethanol administration in rats (Lieber et al., 1989; McBride and Li, 1998) and, as in humans, for long-term continuous enteral nutrition (Muller et al., 1992). This route of administration seems to be associated with less technical problems than continuous i.v. infusion, although it still requires that each rat be connected to an infusion pump via a flexible tubing and swivel, which is not needed when using s.c. implanted osmotic minipumps. At a workshop on model development in epileptogenesis and therapy-resistant epilepsy organized by the NINDS in Gaithersburg, Maryland (November 1, 2006), Ed Betram has recently proposed to use intragastric infusion for long-term administration of AEDs in rodents, but more data are needed to validate this strategy.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

As shown in this review, because of the rapid elimination of most AEDs in rats, it is no trivial task to maintain effective steady-state AED levels in the plasma throughout the day over multiple days. This, however, is needed to ensure that there will be adequate levels in the system for the purpose of the experiment, e.g., at the time the animals may have unpredictable spontaneous seizures. Administration of very high doses of AEDs as used by some groups to overcome this problem may be associated with toxicity and/or unspecific drug effects that may lead to false negative or positive results. Whenever possible, AED levels should be determined during prolonged drug administration to ensure that relevant drug levels were maintained in the experiment. Furthermore, to avoid that maximum tolerable doses are exceeded during treatment, adverse effects of the administered drug should be repeatedly quantified during the period of prolonged treatment, e.g., by assessing muscle impairment by the rotarod test or scoring ataxia and sedation by a rating scale (cf., Hönack and Löscher, 1995). Overall, the use of an adequate dosing regimen, which is based on elimination rate, is an absolute prerequisite when using rat models for discovery of new antiepileptogenic therapies or therapies for pharmacoresistant epilepsy, because otherwise such models may lead to erroneous conclusions about drug efficacy.

In the present review, the elimination rates and effective plasma levels of currently used AEDs were described for rats as a guide for adequate dosing regimens. Fig. 3 provides a step-by-step recommendation for experimenters for selection of an application protocol. In the case of new compounds that are in the drug development pipeline, information on elimination kinetics in rats can usually be obtained from pharmaceutical industry, if these data are not yet published. As shown for PB in this review (Fig. 1), computer modeling is very helpful in developing a suitable dosing regimen for prolonged drug administration when the pharmacokinetics and minimally effective plasma concentrations of a drug are known. The advantages and disadvantages of different application routes for prolonged drug testing in rodents are summarized in Table 4. Before choosing one of these application routes for prolonged drug testing in an epilepsy model, a set of standard control experiments is recommended. These include the development of a suitable drug formulation, assessment of the drug's pharmacokinetics after application of this formulation in the rat strain and gender that will be used for the epilepsy model, assessment of the tolerability of this formulation upon intermittent or continuous administration in rats, and determination of trough and peak plasma levels during prolonged administration (Fig. 3).

image

Figure 3. Step-by-step recommendation for experimenters, planning experiments with prolonged drug administration in rats.

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Table 4. Advantages and disadvantages of different application routes for prolonged AED testing in rodents
Application routesRequirementsAdvantagesDisadvantages
Repeated injections (e.g., i.p., p.o., s.c.)Syringe, appropriate needles. Solubility of drug in tolerable solution, ideally water. Drug suspensions are often be associated with less bioavailability than solutions.Easy to performLarge variation in drug levels between applications; difficult to maintain effective drug levels for drugs with rapid elimination. Too high doses may be associated with toxicity and nonspecific drug effects. Repeated i.p. or s.c. application is often associated with local irritability and inflammation.
Continuous i.v. infusionSyringe, appropriate needles, infusion pump, flexible tubing, swivel. Solubility of drug in tolerable solution, ideally water.Controlled, constant-rate i.v. application of drug allows maintaining any desired concentration in plasma and brain. Bioavailability is 100%.Clotting of the chronically implanted catheters may limit the duration of the experiment, if catheters are not frequently flushed with heparinized solutions. Surgery needed for implantation of catheter.
Continuous intragastric infusion through an indwelling (chronically implanted) gastric tubeSyringe, appropriate needles, infusion pump, flexible tubing. Solubility of drug in tolerable solution, ideally water. Drug suspensions may be associated with less bioavailabilityBoth drug solution and suspensions can be used. Low risk of local irritability in response to drug application. Fewer technical problems than with i.v. infusion.Bioavailability depends on the amount of drug absorbed from the gastrointestinal tract. Absorption may not be dose-dependent and may be retarded. Surgery needed for implantation of catheter.
Prolonged application via drinking waterSolubility of drug in water. Drug should not alter the palatability of the water.Easy to performLarge variation in drug levels because rodents drink mostly during the night. Thus, drug levels during day time (light period) may become subtherapeutic.
Prolonged application via foodDrug should not alter the palatability of the food.Easy to perform; drug needs not to be dissolved.Large variation in drug levels because rodents eat mostly during the night. Thus, drug levels during day time (light period) may become subtherapeutic.
Constant rate application via osmotic minipumpsSolubility of drug in tolerable solution, ideally water. Sufficiently long stability of drug in solution.Convenient method for prolonged drug application.Expensive (minipumps can only be used once). Cannot be used for drugs that are not soluble or stable in solution. Limited reservoir volume of the pump. Surgery needed for implantation of pump.

The future of AED development lies in the discovery of drugs that are antiepileptogenic, disease-modifying, or more efficacious than current AEDs (Löscher and Schmidt, 2006a). For reaching these goals, preclinical drug development is in need of a “paradigm shift” from testing of drugs for anticonvulsant activity after single dose administration in traditional seizure models, such as the maximal electroshock seizure (MES) or pentylenetetrazole (PTZ) tests, to evaluation of chronic drug efficacy in epilepsy models. An important first step in this conceptual evolution is the implementation of animal models of epileptogenesis and pharmacoresistance in drug development. As pointed out in this review, any meaningful drug testing in such models will necessitate the maintenance of effective drug levels throughout a prolonged testing period. Using the principles of drug efficacy testing described in this review may not only improve therapy discovery but also the potential of using drugs as tools to enhance our understanding of the mechanisms underlying epileptogenesis and pharmacoresistance.

Acknowledgments

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES

Acknowledgments:  I thank Prof. M. Kietzmann for calculating maintenance doses of AEDs by the WinNonlin program and Dr. Claudia Brandt for discussions on the manuscript. The author's own studies were supported by the Deutsche Forschungsgemeinschaft (DFG; Bonn, Germany) and a grant (R21 NS049592) from the National Institutes of Health (NIH; Bethesda, MD, U.S.A.).

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTS NECESSITATING PROLONGED DRUG ADMINISTRATION IN RODENT MODELS OF EPILEPSY
  4. ELIMINATION HALF-LIVES OF AEDS IN RATS
  5. EFFECT OF RAT STRAIN AND GENDER ON AED KINETICS
  6. EFFECTIVE AED LEVELS IN PLASMA OF RATS DURING PROLONGED TREATMENT
  7. INFLUENCE OF ADMINISTRATION VEHICLE OR DRUG FORMULATION ON PHARMACOKINETICS OF AEDS
  8. CONVENTIONAL ROUTES OF INTERMITTENT DRUG APPLICATION FOR PROLONGED AED TESTING IN RATS
  9. ROUTES FOR CONTINUOUS AED ADMINISTRATION IN RATS
  10. CONCLUSIONS
  11. Acknowledgments
  12. REFERENCES
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