Phenytoin

Phenytoin (diphenylhydantoin) (Fig. 1) was first introduced as an anticonvulsant agent in 1938, and it is one of the most widely used anticonvulsant drugs. The proposed mechanism of action for phenytoin is to reduce electrical conductance among brain cells, which moderates the runaway brain activity present in seizures. This could be achieved by (a) altering ion fluxes associated with depolarization and/or repolar-ization, (b) altering membrane stability, (c) influencing calcium uptake in presynaptic terminals, (d) influencing the sodium-potassium ATP-dependent ionic membrane pump or a combination of any of those factors. Side effects occurring at blood concentrations

Phenytoin

Fig. 1. Phenytoin.

above the optimum therapeutic interval include sedation, ataxia, and paradoxical seizures. Phenytoin is a low-cost drug with a long history of safe use and as a result it is often a first line of defense for seizure patients. It is important to measure the levels of phenytoin in cases where seizures are not controlled to determine whether blood levels are less than therapeutic or whether the seizures are paradoxical from toxic levels of phenytoin. In addition, phenytoin is highly protein bound (~90%), so in cases where toxicity is suspected but total serum phenytoin is within the optimal therapeutic interval, it becomes important to measure free phenytoin levels.

Early methods for measurement of phenytoin were developed using gas chromatography (GC) (3) and then HPLC on reversed phase columns to assay a panel of anticonvulsant drugs (5,6). In chromatographic approaches, samples are generally mixed with an internal standard, followed by liquid-liquid extraction and then chromatographic analysis. As previously stated, potential interferences in chromatographic methods stem from effects of serum components and other drugs with extraction, or co-eluting substances during chromatography; however, no interferences of this type were reported for phenytoin. A separate approach to measurement of phenytoin involved development of immunoassays based on antibodies directed toward phenytoin. These assays included the enzyme-labeled immunoassays such as enzyme-multiplied immunoassay (EMIT) and cloned enzyme donor immunoassay (CEDIA), and later fluorescence polarization (FPIA), turbidimetric, and chemiluminescent immunoassays.

One of the primary potential interferences in immunochemical measurements of phenytoin (and free phenytoin) is cross-reactivity of the antibodies with the major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH) (Fig. 2) and its glucuronide conjugate (7-12). HPPH is the primary metabolite of phenytoin, and it is readily conjugated to glucuronide (HPPG), which is cleared renally. It is estimated that 60-90% of the administered dose of phenytoin can be recovered in the urine as HPPG (9). This cross-reactivity becomes particularly important in patient with renal insufficiency; as renal clearance of HPPG decreases, the metabolite concentration builds up, and the potential for assay interference increases. Initial studies examining this issue indicated that HPPH and HPPG cross-reactivity in this patient population was not a problem for EMIT-based immunoassays but that the FPIA immunoassay (using TDx analyzer) from Abbott Laboratories, Abbott Park, IL, USA, was affected with respect to both total and free phenytoin measurements (10). In addition, this

Carbamazepine

Fig. 2. 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH).

same study discussed two important points: (a) it mentioned that earlier problems of cross-reactivity with an EMIT-based assay were solved by switching to a monoclonal antibody with lower affinity for the metabolites (suggesting that this would solve the same problem in other assays), and (b) this study postulated from their data that there were additional cross-reactants in the specimens because HPPH and HPPG concentrations could not entirely account for the bias that they were observing with respect to HPLC measurements.

These points are important because a follow-up study by the same group (9) addressed these same points but come to quite different but interesting conclusions. With respect to monoclonal antibodies, it was noted that the TDx assay had been modified (TDx Phenytoin II assay) to utilize a monoclonal antibody reagent. Later, this was discontinued because of cross-reactivity with the drug oxaprozin (In addition, it was noted that oxaprozin seemed to increase measured free phenytoin concentrations by displacement of phenytoin from binding proteins, as well as cross-reactivity with the antibody reagent). As the authors examined their hypothesis that there were additional cross-reactants other than HPPH and HPPG, they found that a different phenomenon was occurring. They were able to demonstrate instead a concentration-dependent cross-reactivity, where the reaction of HPPH and HPPG with the immunoassay reagents was enhanced by increasing concentrations of phenytoin (9). More recent studies have examined cross-reactivity of these metabolites with a chemiluminescent immunoassay on the IMMULITE 2000 from DPC (8) and a turbidimetric immunoassay on the Bayer ADVIA 1650 (7). These studies showed no cross-reactivity of the ADVIA assay with HPPH, and a concentration-dependent cross-reactivity of the IMMULITE assay with HPPH and phenytoin-N-glucuronide (an analog in place of HPPG).

Another drug that must be considered for cross-reactivity with phenytoin assays is fosphenytoin (5,5-diphenyl-3-[(phosphonooxy)methyl]-2,4-imidazolidine-dione disodium salt). In some cases such as treating patients with status epilepticus, or administration of a loading dose for epileptic patients unable to take oral anticonvul-sants, it is desirable to give the drugs through intravenous (IV) or intramuscular (IM) routes of administration. However, phenytoin is poorly soluble in aqueous solution, and it may crystallize in commonly used IV fluids or at the site of IM injection. Fosphenytoin is a phosphate ester derivative of phenytoin that functions as a water-soluble prodrug of phenytoin. This allows the drug to be administered through IV or IM routes. It is rapidly converted to phenytoin after administration (half-life <15 min) and provides the anticonvulsant benefits of phenytoin while avoiding complications associated with parenteral administration of phenytoin. Fosphenytoin is not typically monitored clinically because of its short half-life and lack of pharmacological activity.

Cross-reactivity of fosphenytoin with phenytoin immunoassays has been reviewed in the scientific literature for multiple analytical platforms (8,13-15) (Table 1). Significant cross-reactivity of fosphenytoin in various degrees was found on the TDx phenytoin (14) and phenytoin II (8,13,15), AxSym phenytoin II (13,15), ACS:180 (13,15), Vitros (15), IMMULITE (8), and EMIT 2000 assays (14). The only phenytoin assay that seemed unaffected by the presence of fosphenytoin was the ACA assay from Dade Behring, Newark, DE, USA (8,15). Based on this cross-reactivity, it is recommended that specimens for determination of phenytoin concentrations should not be obtained for patients on fosphenytoin until at least 2 h after IV infusion or 4 h after IM

Table 1

Cross-Reactivity of Fosphenytoin with Phenytoin Immunoassays

Fosphenytoin Fosphenytoin

Assay Cross-Reactivity (%)a Cross-Reactivity (%)b Reference

Table 1

Cross-Reactivity of Fosphenytoin with Phenytoin Immunoassays

Fosphenytoin Fosphenytoin

Assay Cross-Reactivity (%)a Cross-Reactivity (%)b Reference

TDx

32-42

59-75

13

TDx-II

>250

>250

13

Axsym-II

17-29

30-78

13

ACS:180

48-52

50-51

13

ACA Star

8-14

2-8

15

TDX-II

518

Not reported

15

Vitros

6-7

2-5

15

TDx

22-120

32-451

14

EMIT 2000

7-13

-20 to 50

14

IMMULITE 2000

64

Not reported

8

a Cross-reactivity in the absence of phenytoin. b Cross-reactivity in the presence of phenytoin.

a Cross-reactivity in the absence of phenytoin. b Cross-reactivity in the presence of phenytoin.

injection. Also, incubating 1 ml specimen with 10 ^l of alkaline phosphatase enzyme (Sigma Chemical Company, St. Louis, MO, USA) converts any fosphenytoin present in the specimen to phenytoin within 5 min at room temperature. This procedure eliminates interference of fosphenytoin in phenytoin immunoassays. The authors observed complete conversion of fosphenytoin to phenytoin by alkaline phosphatase in heparin, ethylenediaminetetraacetic acid (EDTA), and citrated plasma (16).

Roberts et al. (15) studied in detail falsely elevated phenytoin values when measured by immunoassays compared with HPLC in patients with renal failure. The authors observed falsely increased phenytoin results up to 20 times higher than the HPLC results using AxSYM, TDx Phenytoin II (Abbott Laboratories, Abbott Park, IL), ACS:180 (Bayer Diagnostics, Tarrytown, NY), and Vitros assays. The ACA star results were comparable to HPLC values. Interestingly, no fosphenytoin was detected in any of these specimens by using HPLC. For example, in the renal failure patient 3 on the 9th day of the hospital stay (300 mg of fosphenytoin dosage), the phenytoin concentration as measured using the HPLC was 5.3 ^g/mL. The corresponding phenytoin concentrations measured by immunoassays were 6.3 (ACA Star), 22.0 (ACS:180), 12.7 (AxSYM), and 28.0 ^g/mL (TDxII) respectively. On the basis of their study with several patients, the authors proposed the presence of a novel metabolite of fosphenytoin, which has a very high cross-reactivity with antibodies, used in several immunoassays for phenytoin (15). Later, Annesley et al. identified a unique immunore-active oxymethylglucuronide metabolite derived from fosphenytoin in sera of uremic patients and explained the mechanism of falsely elevated phenytoin in these patients with uremia receiving fosphenytoin (17).

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