Adsorption of Fluoxetine HCl by Activated Charcoal

NOTES

Adsorption of Fluoxetine HCl by Activated Charcoal DAVID O. COONEYX

AND

ROBERT THOMASON

Received October 8, 1996, from the Department of Chemical Engineering, University of Wyoming, Laramie, WY 82071. manuscript received February 5, 1997. Accepted for publication February 7, 1997X. Abstract 0 We studied the adsorption of fluoxetine HCl (Prozac) by Norit USP XXIII activated charcoal in vitro, in simulated gastric fluid (USP; pH 1.2), and in simulated intestinal fluid (USP; pH 7.5). The data were fitted to both Langmuir and Freundlich equations. The Langmuir Qm values (maximal adsorption capacities) for pH 1.2 and 7.5 were 0.258 and 0.330 g drug/g charcoal, respectively. These excellent capacities suggest that oral charcoal therapy would be effective for fluoxetine overdose.

Introduction Fluoxetine HCl (Prozac) is reported to have oral LD50 values of ∼450 and 250 mg/kg in rats and mice, respectively. The acute lethal dose in humans has not been established. Moderate fluoxetine overdoses cause a variety of adverse reactions, such as agitation, hypomania, tremor, and other signs of central nervous system (CNS) excitation. Acute overdoses can cause nystagmus, nausea, vomiting, coma, tachycardia, and ECG changes.1 Several fatalities following fluoxetine overdosage have been reported.1 In one case, a patient ingested 1.8 g of fluoxetine and an unknown amount of maprotiline, and in another case, a patient ingested fluoxetine and alcohol concomitantly and then died. A few deaths have occurred as a result of fluoxetine alone. Treatment of fluoxetine overdose in humans normally involves symptomatic and supportive care. There is no specific antidote for fluoxetine. Although administration of oral activated charcoal has been useful in the treatment of acute overdosage with tricyclic antidepressants,2 the effect of oral activated charcoal on the absorption of fluoxetine from the gut is not currently known. To establish a rationale for oral charcoal therapy in fluoxetine overdose, in vitro studies of fluoxetine adsorption by a USP activated charcoal were performed.

Materials and Methods Fluoxetine HCl, reference standard grade, was supplied by Eli Lilly, Inc. (Indianapolis, IN). It has a formula weight of 345.76, and its pKa is 9.5-9.8, as determined by Eli Lilly in an aqueous 66% dimethylformamide solution (the choice of this medium is unexplained).3 We have estimated the pKa of this drug as ∼9.1 by measurement of the pH in a 0.01 M aqueous solution. A stock solution (1 g/L; 2.89 mM) of fluoxetine HCl was prepared in simulated gastric fluid USP, pepsin omitted (SGF), which consists of 2.0 g/L NaCl and 7 mL/L of 12N HCl, and has a pH of 1.2. Daly and Cooney4 have shown that the omission of pepsin from SGF has only a small effect on the in vitro adsorption of drugs. X

Abstract published in Advance ACS Abstracts, April 1, 1997.

642 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997

Final revised

If a person were to ingest 50 20-mg Prozac capsules (the only size marketed) and these were to mix with 1 L of stomach fluid, a drug concentration of 1 g/L would result. Thus, 1 g/L is in the range of an expected overdose. As a point of reference, the solubility of fluoxetine HCl in water is on the order of 14 g/L.3 Thus, the drug was completely dissolved in our stock solutions. An ultraviolet (UV) absorption scan was run on a sample of the stock, diluted with SGF, and a peak at 226 nm was identified as suitable for use in assaying the concentration of fluoxetine in subsequent samples. Scans of the diluted stock were run daily for several days to monitor drug stability, and no changes were noted. Various amounts of Norit USP XXIII activated charcoal (American Norit Company, Atlanta, GA), which has an internal surface area (BET method) of 900 m2/g, were weighed into new 25-mL capacity glass scintillation vials, 20 mL of the stock was pipetted into each, and the vials were capped. The vials were placed in a shaker overnight (30 min is sufficient for equilibrium, as shown by Cooney and Kane5) and the mixtures were filtered through 13-mm diameter cellulose nitrate filters (Millipore 0.45-µm pore-size, type HAWP) held in stainless steel filter holders. The first 5-10 mL were discarded, and the next 2-3 mL were collected in a second set of new scintillation vials. Then, 0.2 mL of each clear filtrate was pipetted into a third set of new scintillation vials, into which 20 mL of SGF had been pipetted. The absorbances of these diluted samples at 226 nm were determined in a Perkin Elmer Lambda 9 spectrophotometer. A stock “blank”, which was treated in the same way as the samples with respect to overnight shaking, filtration, and dilution, had an absorbance of 0.3579. Although the spectrophotometer read out values on a digital display to a resolution of ( 0.0001, immediate triggering of the “read” button on the unit one or more times, without touching the sample, gave additional values which differed by about ( 0.0005. This variation is of little consequence. All absorbance values fell into the Beer’s Law range, where absorbance and concentration are strictly proportional. Linearity was established by preparing stock solutions of 0, 0.2, 0.4, 0.6, and 0.8 g/L by careful pipetting, diluting these as indicated, and measuring their absorbances. These values, along with that for the 1.0 g/L stock, were plotted versus concentration. Linear regression indicated linearity with an R2 (correlation coefficient) of >0.998. The sensitivity of the assay procedure was estimated to be ∼0.0005 absorbance units. Thus, the minimum measurable concentration, distinguishable from a blank sample, would be ∼0.0014 g/L. Precision of the assay was evaluated by running several assays of selected samples, and these gave absorbance readings which were within ( 0.002 of each other, or about ( 0.006 g/L concentration. For the purposes of this study, which was to indicate in general how well fluoxetine HCl adsorbs to a representative charcoal, the assay procedure was more than satisfactory. The residual fluoxetine concentrations (Cf) were determined by dividing the sample absorbances by 0.3579 and multiplying by 1 g/L. Finally, the amount of drug adsorbed on the charcoal was calculated from the mass balance equation Q ) V(Co - Cf)/W, where V is the volume of solution (0.020 L), Co is the initial concentration (1 g/L), Cf is the final concentration (in g/L), and W is the weight of charcoal (g). Thus Q ) g drug adsorbed/g charcoal. The Q versus Cf values were plotted to give the adsorption “isotherm” (all tests were conducted at room temperature, 20-21 °C). The data were fit by linear regression to the Langmuir isotherm equation Q ) KQmC/(1 + KC) using a plot of 1/Q versus 1/Cf. On such a plot, the intercept is 1/Qm and the slope is 1/KQm; thus, one

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Figure 1sAdsorption isotherms for fluoxetine.

Figure 2sPlots for determining Langmuir parameters.

can determine the two parameters K and Qm from the intercept and slope values. The Qm is the maximal or asymptotic value of Q as Cf f ∞. The data were also plotted on log-log coordinates for fitting by linear regression to the Freundlich equation, Q ) KCf1/n, which was expressed in linear form as log Q ) log K + (1/n) log Cf. Thus, from the slope on such a plot and the value of Q at Cf ) 1 (for which log Cf ) 0, and the equation reduces to log Q ) log K), one can determine the two parameters K and 1/n. The entire procedure was repeated using a stock solution of 1 g/L fluoxetine HCl in simulated intestinal fluid USP, pancreatin omitted (SIF), which consists of 6.8 g/L K2HPO4 and 1.52 g/L NaOH, and has a pH of 7.5. The stock sample absorbance at 226 nm was 0.3648. Stability of the drug at this pH was monitored by running scans on the diluted stock daily for several days. No changes were noted. Assay linearly, instrument precision and reproducibility, and variation of absorbance values on duplicate samples of the diluted filtrates were the same as in the pH 1.2 case.

Results and Discussion The adsorption isotherms of fluoxetine in SGF and SIF in terms of Q versus Cf are shown in Figure 1. Fluoxetine HCl is a basic drug so it is protonated (positively charged) at low pH. As the pH increases, it becomes progressively less ionized. However, using the Henderson-Hasselbalch equation and a pKa value of 9.1, it is still 97.55% ionized at pH 7.5. Neutral forms of molecules are known to adsorb much better to activated charcoal than their ionized counterparts because adjacently adsorbed neutral molecules do not electrostatically repel each other. Cooney and Wijaya6 have demonstrated this kind of pH effect clearly for a wide variety of acidic, basic, and amphoteric substances adsorbing onto activated charcoal. However, the differences in adsorption shown in Figure 1 for pH 7.5 and pH 1.2 are much larger than having 2.45% of the drug in neutral form would suggest. The ionic strength of the pH 7.5 medium is 0.136 M, or somewhat greater than the 0.116 M ionic strength of the pH 1.2 medium. This higher ionic strength, or perhaps a greater effectiveness of PO42- than Cl- in reducing electrostatic interactions between adjacently adsorbed fluoxetine molecules, may account for the better adsorption at pH 7.5. Cooney and Wijaya6 have also demonstrated an ionic strength effect due to added salts quite clearly for the adsorption of largely ionized molecules. Langmuir parameters were determined by plotting 1/Q versus 1/Cf. However, values at high 1/Cf (i.e., low Cf) were

Figure 3sFreundlich plots of fluoxetine data.

far from linear on such a plot, and only values for low 1/Cf, corresponding to Cf values greater than ∼0.1 g/L, were used. This means that we attempted to obtain a good fit to the isotherm for Cf > 0.1, in exchange for a poorer fit for Cf < 0.1. One can not fit the entire Cf range well. As shown in Figure 2, even the data employed do not give a strong linear fit. The Langmuir parameters obtained were: For SGF, K ) 50.68 L/g and Qm ) 0.258 g/g, and for SIF, K ) 35.02 L/g and Qm ) 0.330 g/g. Thus, the Langmuir equations are Q ) 13.08 Cf/(1 + 50.68 Cf) for SGF (pH 1.2) and Q ) 11.56 Cf/(1 + 35.02 Cf) for SIF (pH 7.5). The Qm values are important, because they indicate the maximal amounts that can be adsorbed when all available adsorption sites are occupied. The Qm values of 25.8 and 33.0 wt% are quite good. The Freundlich plots are shown in Figure 3. As often occurs for the adsorption of organics from aqueous solution, excellent straight-line relationships were obtained. The best-fit equations are Q ) 0.259Cf0.0696 for SGF (pH 1.2), and Q ) 0.322 Cf0.0558 for SIF (pH 7.5). The exponent values (0.0696, 0.0558) indicate very strong adsorption at low surface coverage, as one can see from the very steep curves in Figure 1 at low concentrations.

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of organic species from aqueous solutions (e.g., organic contaminants from wastewaters) have shown a similar superiority of the Freundlich equation over the Langmuir equation for fitting equilibrium data.

Conclusions

Figure 4sComparison of data (b), Langmuir equation (4), and Freundlich equation (0) for the SGF case.

Charcoals with higher surface areas, such as Norit B Supra (surface area, 1400 m2/g) and Norit A Supra (surface area, 2000 m2/g), would likely perform much better, as the results of Cooney7 have shown for other drugs. A comparison of the data, the best-fit Langmuir equation, and the best-fit Freundlich equation for the SGF (pH 1.2) case are shown in Figure 4. The Langmuir equation does not fit the data closely at low concentrations, because we had to sacrifice a good fit there to achieve a good fit at higher concentrations, as already mentioned. The Freundlich equation fits extremely well over the entire range. It may be noted that the Langmuir equation predicts a plateau in Q (equal to Qm) as Cf becomes large, whereas the Freundlich equation indicates that Q continually rises as Cf increases. Yet, it is obvious that in reality there must be an upper limit to Q. Thus, it is clear that the Freundlich equation must ultimately fail at sufficiently high Cf values. However, in the Cf range of this study, this limit is not approached. A very large number of studies of the adsorption

644 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997

Our in vitro results show that fluoxetine HCl adsorbs very well to Norit USP XXIII activated charcoal and adsorbs better at pH 7.5 than at pH 1.2. The Langmuir Qm values (maximal adsorption capacities) for pH 1.2 and pH 7.5 were 0.258 and 0.330 g drug/g charcoal, respectively. There is every reason to expect that oral activated charcoal therapy would be effective for fluoxetine overdose. Fluoxetine requires 4-8 h for peak plasma concentrations to develop with therapeutic doses, and likely significantly longer in the case of overdoses. Thus, the rate of absorption of fluoxetine is not especially rapid, and this would allow for charcoal to effectively bind the drug if the charcoal were given without undue delay. Clearly, in vivo tests are needed to establish the efficacy of oral charcoal therapy.

References and Notes 1. AHFS Drug Information ‘96; American Hospital Formulary Service, American Society of Hospital Pharmacists: Bethesda, MD, 1996; pp 1601-1602. 2. Cooney, D. O. Activated Charcoal in Medical Applications; Marcel Dekker: New York, 1995, 222-226, 325-327. 3. Gonzalez, J., Eli Lilly, Inc., Indianapolis, IN, private communication, 1996. 4. Daly, J. S.; Cooney, D. O. J. Pharm. Sci. 1978, 67, 1181-1183. 5. Cooney, D. O.; Kane, R. P. Clin. Toxicol. 1980, 16, 123-125. 6. Cooney, D. O.; Wijaya, J. In Proceedings, Second International Conference on the Fundamentals of Adsorption; Liapis, A., Ed.; Engineering Foundation: New York, 1987; pp 185-194. 7. Cooney, D. O. J. Toxicol. Clin. Toxicol. 1994, 33, 213-217.

Acknowledgments This study was supported by a grant from Eli Lilly, Inc., Indianapolis, IN.

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