METABOLITES OF THE ANTIPSYCHOTIC AGENT CLOZAPINE INHIBIT THE REPLICATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

Abstract

Schizophrenia is a serious and often debilitating neuropsychiatric disease of worldwide importance. Current therapy relies on the use of typical antipsychotic medications, which specifically inhibit binding of ligand at the D2 dopamine receptor, and atypical medications which display little activity for this receptor interaction. While atypical antipsychotic agents have been shown to variably inhibit other neuroreceptor-ligand interactions, the exact mechanisms for the therapeutic efficacy of these medications have not been completely defined. Clozapine, an atypical antipsychotic, and nine of its metabolites were studied in vitro for possible antiviral activity against a model of a human neurotropic virus, human immunodeficiency virus type (HIV-1). In an assay for inhibition of virus-induced cytopathic effect (CPE) two metabolites demonstrated antiviral activity (ID₅₀ – 37-85m M ), while other atypical or novel antipsychotics as well as typical medications had no effect. Based on an ELISA, four chemically similar metabolites inhibited the production of p24, the major internal antigen of HIV (ID₅₀ = 11.6-15.7m g/ml) (38-51 m M). These data suggests that the therapeutic efficacy of some antipsychotics may be due in part to an ability to inhibit viral replication. Antiviral agents may prove to be effective adjuncts in the treatment of schizophrenia.

Keywords: Antipsychotic; Antiviral; Schizophrenia; Virus

1. Introduction

Schizophrenia is a serious neuropsychiatric disease of worldwide importance which causes a high degree of morbidity. It is also an extremely expensive disease, with direct and indirect costs in the United States for 1991 estimated at $65 billion (Wyatt et al. 1995). Antipsychotic medications constitute the principal form of therapy for the management of patients with schizophrenia. Many of the traditional or typical antipsychotics are members of the phenothiazine class of compounds, which are potent antagonists of the D2 form of dopamine receptors within the central nervous system, and have been thought to exert their antipsychotic effect predominantly by the inhibition of the biding of the ligand (Leysen et al., 1994; Glatt et al., 1995). However, the use of these typical antipsychotics is associated with a range of extra-pyramidal side effects related to the inhibition of D2 receptor binding (Leysen et al., 1994; Glatt et al., 1995). For this reason other classes of medications have been developed for the treatment of patients with schizophrenia.

The first effective atypical antipsychotic available for the treatment of patients with schizophrenia was the heterocyclic compound clozapine. Clinical trials have indicated that clozapine is an effective antipsychotic and it has not been associated with a high degree of extra-pyramidal side effects (Cheng et al., 1988; Baldessarini and Frankenburg, 1991; Perry et al., 1991; Jann et al., 1993; Centorrino et al., 1994; Glatt et al., 1995). However, its clinical utility has been limited by the occurrence of agranulocytosis in some individuals taking the medication (Baldessarini and Frankenburg, 1991; Jann et al., 1993). Clozapine has been shown to inhibit the binding of ligand to a number of neural receptors, including the D4 and 5HT receptors. Clozapine also binds to other neural receptors and the precise mechanism by which clozapine exerts its antipsychotic effect has not been established (Hartvig et al., 1986; Baldessarini and Frankenburg, 1991; Jann et al., 1993; Glatt et al., 1995).

Previous studies of typical antipsychotics and other neurotropic agents have indicated that many of them possess antimicrobial properties as evidenced by their ability to inhibit the replication of a range of viruses and other microbial organisms (Libikowa et al., 1977; Pearson et al., 1982; Bohn et al., 1983; Nemerow and Cooper, 1984; Patou et al., 1986; Kristiansen et al., 1991). Some typical antipsychotics display apparent inhibition of retrovirus replication (Wunderlich and Sydow, 1980; Wunderlich et al., 1980; Wunderlich and Zotter, 1982; Corbascio and Bufalini, 1989), a finding which is of particular interest in light of the recent demonstration of reverse transcriptase activity in the brains of some individuals with schizophrenia (Jones-Brando et al., 1995). The characterization of antiviral activity in a range of antipsychotics might indicate a possible mechanism of action and provide the rationale for the development of new antipsychotic agents. We performed experiments to determine if atypical antipsychotics including clozapine, risperidone, seroquel, and sertindole have antiviral activity against human immunodeficiency virus type 1 (HIV-1), a known neurotropic virus causing disease in the CNS (Ayuso, 1994) in defined cellular replication systems.

1. Methods

2.1 Cells and viruses

The human T-lymphocyte cell lines CEM-T4 (Foley et al., 1965) and MT-2 (Harada et al., 1985) were grown in RPMI-1640 medium containing 25 mM Hepes and supplemented with 10% fetal bovine serum, 2 mM L-Gln, 50 units of penicillin G per ml, and 50m g of streptomycin sulfate per ml. Primary cultures of human peripheral blood leukocytes (PBLs) were grown in medium containing equal parts of RPMI-1640 (containing 25 mM Hepes and supplemented with 2 mM L-Gln) and Dulbecco’s Modified Eagle Medium (containing 25 mM Hepes and high glucose), supplemented with 10% heat-inactivated human AB serum (Advanced Biotechnologies; Columbia, MD), 50 units of penicillin G per ml, 50 m g of streptomycin sulfate per ml, and 1.0 unit of recombinant human interleukin-2 (AIDS Research and Reference Reagent Program) per ml. All media components were purchased from Life Technologies Inc. (Gaithersburg, MD) unless otherwise noted.

The Isolates of HIV-1 used in these experiments were the standard laboratory strain HTLV-III_B, a clinical isolate, designated H112-2, having only limited passage on MT-2 cells (Larder et al., 1989), and a freshly isolated clinical sample, designated 16, passaged only twice on PBLs prior to use.

2. 2. Pharmacological compounds

Clozapine and nine of its metabolites (see Fig. 1) were provided by Sandoz Pharmaceuticals Corp. (East Hanover, NJ). Risperidone and 9-OH-risperidone were provided by Janssen Research Foundation (Beerse, Belgium). Seroquel and sertindole were provided by Zeneca Pharmaceuticals (London, UK) and H. Lundebeck A/S (Copenhagen, Denmark), respectively. Lithium carbonate, haloperidol, fluphenazine, thiothixene, chlorpromazine, and 2’,3’-dideoxyinosine (DDI) were purchased from commercial sources. 3’-Azido-3’-decxythymidine (AZT) was obtained from the NIAID AIDS Research and Reference Reagent Program. All test compounds were evaluated within a range of 0.625-200 m g/ml final concentration, corresponding to 1-680 m M, except for 7-hydroxy-8-deschloro- and 2-hydroxy-8-deschloro-clozapine (range+6.25-400 m g/ml for 20-1290 m M) and lithium carbonate (range=6.25-200 m g/ml for 85-2700 m M).

 

2. 2. CPE-inhibition assay
   3. Antiviral activity was measured as the inhibition of viral cytopathogenic effect (CPE). Test cells were treated with polybrene (2 m g/ml) for 30 min at 37^oC. After treatment, 1 x 10⁴ cells were dispensed into each well of a 96-well tissue culture tray (Costar). A 50 m l volume of each test compound dilution (prepared as a 4 x concentration) was added to five wells of cells, and then the cells were incubated at 37^oC for 1 h. Virus stock was diluted in cell culture medium and added to three of the wells for each test compound concentration (final multiplicity of infection of 0.05 with HTLV-III_B and 0.1 with H112-2). Culture medium without virus was added to the remaining two wells of each drug concentration to allow for the evaluation of cytotoxicity.Assay plates were incubated at 37^oC in a 5% CO₂ atmosphere and examined microscopically each day for CPE. When the virus control samples displayed maximal CPE, the surviving cells in each assay well were quantified using the MTT assay (Pauwels et al., 1988; Schwartz et al., 1988). For compounds displaying a dose-dependent effect for either CPE-inhibition or cytotoxicity, values for the median inhibitory dose (ID₅₀) and median toxicity dose (TD₅₀), respectively, were calculated using the dose-effect analysis software of Chou and Chou (1987).
   4. p24 antigen inhibition assay

Peripheral blood from HIV-negative donors was collected in heparinized tubes and processed within 4 h of collection. PBLs were obtained by discontinuous density gradient centrifugation using Lymphocyte Separation Medium (Organon Teknika; Durham, NC), then thoroughly washed, suspended at a density of 1.5 x 10⁶ cells per ml in culture medium, and grown for 3 days in the presence of phytohemagglutinin (PHA-P, 10m g/ml; Sigma). After the 3-day PHA stimulation, PBLs were treated with polybrene, concentrated by low speed centrifugation, and then suspended in virus inoculum (isolate 16; final moi=0.01) at a cell concentration of 2-4 x 10⁶ cells per ml. The cells were incubated for 2 h at 37^oC with occasional mixing, diluted and then washed to remove unabsorbed virus. The cells were then resuspended in fresh medium, and 2 x 10⁵ cells were dispensed into each well of a 24-well tissue culture plate. Mock infected cells were similarly processed and dispensed. Drug dilutions (prepared as 2 x concentrations) were added to two wells of infected cells and one well of uninfected cells, and the assay plates were incubated at 37^oC in a 5% CO₂ atmosphere.

An aliquot of each supernatant was collected on the fifth day post-infection and tested for HIV p24 antigen by enzyme immunoassay (Dupont; Boston, MA). For each dilution of test compound, the percentage inhibition of p24 was calculated relative to the amount of p24 generated in untreated virus-infected cells. The ID₅₀ and ID₉₀ values were determined using the dose-effect analysis software of Chou and Chou (1987). Cytotoxicity was determined after 4 days of drug treatment by reaction of uninfected, drug-treated cells with MTT as described above.

2. Results

We evaluated the atypical antipsychotics and metabolites for their ability to inhibit the CPE of two different strains of HIV in MT-2 and CEM-T4 cells. As depicted in Table 1, we found the 8-OH-deschloro-clozapine and 8-OH-desmethyl-clozapine displayed clear inhibition of HIV in both cell lines at concentrations which were not cytotoxic. The ID₅₀ of 8-OH-deschloro-clozapine was 56 m g/ml (182 m M) in MT-2 cells and 37 m g/ml (119 m M) in CEM-T4 cells. The ID₅₀ of 8-OH-desmethyl-cloapine was 85 m g/ml (289 m M) in MT-2 cells and 37 m g/ml (126 m M) in CEM-T4 cells. The metabolite clozapine-N-oxide displayed some CPE inhibition in MT-2 cells. In subsequent repeat experiments, this compound also showed limited antiviral activity in CEM-T4 cells (data not shown). All three of these compounds were determined non-cytotoxic at concentrations < 100 m g/ml.

Table 1
Antiviral evaluation of antipsychotics

Compound name	MT-2		CEM-T4
	IDa50	TDb50	ID50	TD50
Clozapine	—c	17	—	21
N-desmethyl-clozapine	—	7	—	11
Clozapine-N-oxide	81	>100	—	>100
8-OH-8-deschloro-clozapine	56	>100	37	>100
8-OH-desmethyl-clozapine	85	>100	37	>100
Thiomethyl-desmethyl-clozapine	—	17	23	28
2-OH-8-deschloro-clozapine	NDd	ND	—	181
7-OH-8-deschloro-clozapine	ND	ND	—	173
8-thiomethyl-clozapine	ND	ND	—	28
2-OH-clozapine	ND	ND	—	18
Risperidone	—	15	—	107
9-OH-risperidone	—	24	—	134
Sertindole	—	3.5	—	17
Seroquel	—	17	—	68
Haloperidol	—	>20	—	132
Fluphenazine	—	4.7	—	9.1
Thiothixene	—	5.6	—	8.4
Chlorpromazine	—	5.3	—	6.3
Lithium carbonate	—	>200	—	>200
AZT	0.02	38	ND	ND
DDI	ND	ND	0.43	48

Test compounds were added to MT-2 and CEM-T4 cells. The cells were then infected with HIV strain HTLV-III_B (CEM-T4 cells), strain H112-2 (MT-2 cells), or left uninfected. When virus control wells displayed maximal CPE, the number of surviving cells in each well was determined using the MTT assay (Pauwels et al., 1988; Schwartz et al., 1988). Values for the 50% effective dose (ID₅₀ and TD₅₀) were calculated using the dose-effect analysis software of Chou and Chou (1987).
^aMedian inhibitory dose (m g/ml), a measure of CPE-inhibition.
^bMedian toxicity dose (m g/ml), a measure of cytotoxicity.
^cUnable to determine an ID₅₀ due to either cytotoxicity or lack of CPE-inhibition.
^dNot done.

The metabolite thiomethyl-desmethyl-clozapine displayed some inhibition of HIV CPE in CEM-T4 cells but the ID₅₀ (23 m g/ml) (71 m M) was very close to the TD₅₀ (28 m g/ml) (86 m M). The parent compound clozapine did not display specific antiviral activity but was 100% cytotoxic at concentrations of > 50 m g/ml (153 m M).

Significant levels of HIV CPE inhibitory activity were not found for the other four clozapine metabolites, risperidone, 9-OH risperidone, sertindole, seroquel, haloperidol, fluphenazine, thiothixene, chlorpromazine, or lithium carbonate. However, with the exception of lithium, all of these compounds were cytotoxic for MT-2 cells at concentrations above 24 m g/ml. Additionally, more than half of these compounds were toxic to CEM-T4 cells (Table 1).

The 8-OH-deschloro-, 8-OH-desmethyl-, 2-OH-deschloro-, and 7-OH-deschloro- metabolites of clozapine were further evaluated for cytotoxicity and for the ability to inhibit the replication of a fresh clinical isolate of HIV in human PBLs (Table 2). All four compounds had similar inhibitory effects on HIV replication, with calculated ID₅₀ and ID₉₀ values of 15.4 m g/ml (50 m M) and 56.3 m g/ml (183 m M) for 8-OH-8-deschloro-clozapine, 11.9 m g/ml (40 m M) and 30 m g/ml (101 m M) for 8-OH-8-desmethyl-clozapine, 11.6 m g/ml (38 m M) and 32.3 m g/ml (106 m M) for 2-OH-8-deschloro-clozapine, and 15.7 m g/ml (51 m M) and 35 m g/ml (114 m M) for 7-OH-8-deschloro clozapine. 8-OH-8-deschloro-clozapine was the least cytotoxic to PBLs for the four metabolites tested with a TD₅₀ of 56.4 m g/ml (183 m M). The antiviral activities for control compounds AZT and DDI were also measured in this assay, with values for AZT (ID₅₀=2.3 nM; ID₉₀=13.9 nM) and DDI (ID₅₀=0.69 m M; ID₉₀=2.87 m M) consistent with previous studies.

Table 2
HIV p24 inhibition by clozapine metabolites

Compound name	IDa50 (m g/ml)	IDb50 (m g/ml)
8-OH-8-Deschloro-clozapine	15.4 (50 m M)	56.3 (183 m M)
8-OH-Desmethyl-clozapine	11.9 (40 m M)	30.0 (101 m M)
2-OH-8-Deschloro-clozapine	11.6 (38 m M)	32.3 (106 m M)
7-OH-8-Deschloro-clozapine	15.7 (51 m M)	35.0 (114 m M)

PHA-stimulated PBLs were infected with a freshly isolated clinical strain of HIV-1 (strain 16) and then treated with four metabolites of clozapine or left untreated. Supernatant culture medium was collected 5 days after infection, and the HIV-1 p24 core antigen content was determined by ELISA. ID₅₀ and ID₉₀ values were calculated using the dose-effect analysis software of Chou and Chou (1987).
^aDose which inhibited 50% of p24 production relative to untreated control.
^bDose which inhibited 90% of p24 production relative to untreated control.

2. Discussion

These studies document that metabolites of the antipsychotic drug clozapine inhibit the replication of HIV in tissue culture. HIV inhibition was noted with three strains of virus and in three tissue culture systems, indicating that the inhibition is not limited to laboratory-adapted strains of virus. Furthermore, the inhibition occurred at concentrations of the metabolite which were not cytotoxic, indicating that the observed antiviral effect cannot be attributed to cellular destruction.

We did not detect anti-HIV activity in the parent drug clozapine. This finding may indicate that the drug may need to be metabolized in order to develop such activity. However, since clozapine was cytotoxic at concentrations as low as 17 m g/ml (52 m M), it is also possible that anti-HIV activity could not be measured due to cytotoxicity. Similarly, while we did not detect anti-HIV activity in the other atypical antipsychotics, it is possible that less cytotoxic metabolites of these compounds might also display anti-HIV activity.

We also detected an apparent structural-functional relationship between the chemical structure of the clozapine metabolites and corresponding antiviral and cytotoxic activity. Thus the metabolites with the most potent anti-HIV activities contained an hydroxyl group somewhere on the ring structure and lacked the chloride at the 8 position. These compounds were also the least cytotoxic as evidenced by the fact the cells were not altered by concentrations as high as 100 m g/ml (290-340 m M). The reasons for this structural-functional interaction are not known with certainty, but may be related to an alteration of the pK_a of the compounds affected by these substitutions. In any case, these studies indicate that the analysis of additional metabolites of clozapine and the other atypical antipsychotics may yield compounds with higher degrees of antiviral activity and lower degrees of cytotoxicity.

The OH derivatives of clozapine were capable of inhibiting the replication of a wild-type strain of HIV at concentrations between 11.6-15.7 m g/ml (38-51 m M). For comparison, the therapeutic steady-state plasma concentration of clozapine is thought to be 350 ng/ml (Perry et al., 1991; Jann et al., 1993; American Society of Health-System Pharmacists, 1996). However, with large interindividual variations, the range of plasma or serum concentrations of clozapine reported in patients undergoing clozapine treatment is quite wide (60-2121 ng/ml) (Cheng et al., 1988; Baldessarini and Frankenburg, 1991; Perry et al., 1991; Jann et al., 1993; Nordström et al., 1995). Reports of the plasma/serum concentrations of the metabolites of clozapine vary, but more agree that the level of N-desmethyl clozapine is 90-93% that of clozapine. Based on the plasma concentrations above, that would be ~54-1973 ng/ml. Depending upon the measurement technique, the N-oxide metabolite may be present in serum in 10-26% (~6-551 ng/ml) the level of the parent drug (Breyer and Villumsen, 1976; Centorrino et al., 1994). The plasma concentrations attained by the hydroxy metabolites are unknown or have not been reported.

Perhaps more important than plasma concentration is the concentration of parent drug and metabolites in the brain. Unfortunately, the distribution and concentrations of clozapine and its metabolites in the central nervous system (CNS) in humans has not been characterized, and there is reportedly little or no correlation between plasma drug concentration and clinical benefits (Baldessarini and Frankenburg, 1991; Jann et al., 1993). Nordström et al. (1995) found that plasma concentration of clozapine did not predict the degree of D₁, D₂, and 5-HT₂ receptor occupancy in brains of schizophrenic patients. Animal studies show that, in monkeys, clozapine quickly (5-12 min) reaches high levels in certain regions of the brain (Hartvig et al., 1986). In mice and rats, clozapine is distributed widely to many organs in addition to the brain and achieves concentrations in these tissues up to 50 times that in the blood (American Society of Health-System Pharmacists, 1996). The range of concentrations tested in our experiments only captures the upper limits of reported plasma levels of clozapine and two of its metabolites. The ID₅₀ values of p24 inhibition (11.6-15.7 m g/ml) (38-51 m M) are 6-8 times higher than the peak reported for N-desmethyl clozapine and 21-28 times higher than that for clozapine-N-oxide. These may be achievable concentrations in the brain. Finally, it is not possible to strictly compare concentrations in a cell culture well to those in the bloodstream of a complex organism such as a human.

The range of ID₅₀ values which we have found effective against HIV-1 in vitro is higher than that of currently approved anti-retroviral reverse transcriptase antagonists such as AZT and DDI (see Section 3). These levels indicate that the metabolites of clozapine which we examined are unlikely to be useful for the

treatment of systemic infections with HIV-1. Initial experiments to determine the mechanism of the antiviral activity indicate that these metabolites do not inhibit the viral reverse transcriptase (Jones-Brando, unpublished data). However, the fact that, in animal experiments, antipsychotic medications may reach high concentrations within the central nervous system (Hartvig et al., 1986) suggests that these or similar compounds may prove to be useful for the treatment of central nervous system infections with neurotropic viruses (Ayuso, 1994). It is also possible that neurotropic viruses from other genuses will be inhibited by lower concentrations of these medications. For example, preliminary studies indicate that clozapine metabolites can also inhibit the in vitro replication of Borna disease virus (Jones-Brando et al., 1996).

Our findings are consistent with previous studies which have documented antimicrobial activities in typical antipsychotic medications. For example, several phenothiazine and thiothixene compounds inhibit the replication of herpes simplex virus types 1 and 2 (Patou et al., 1986; Kristiansen et al., 1991), tick-borne encephalitis virus (Libikowa et al., 1977), Epstein-Barr virus (Nemerow and Cooper, 1984), and measles virus (Bohn et al., 1983). Two murine retroviruses reportedly are inhibited by reserpine, a weak antipsychotic (Wunderlich and Zotter, 1982), and haloperidol (Wunderlich et al., 1980). Furthermore, the administration of chlorpromazine has been associated with the suppression of genital infections with herpes simplex virus (Chang, 1975) and an apparently slower progression to AIDS in HIV-infected patients (Corbascio and Bufalini, 1989). There are also suggestions that liquid fluphenazine, a phenothiazine, can suppress oral herpes simplex disease (Torrey, 1988). In addition, the antidepressant medication lithium can inhibit the replication of herpes simplex viruses, pseudorabies virus, and vaccinia virus (Skinner et al., 1980; Ziaie and Kefalides, 1989) and the administration of lithium compounds has been associated with the suppression of oral and genital herpes infections (Lieb, 1979; Skinner, 1983; Amsterdam et al., 1990a, b). These findings indicate that antiviral activity may be a common property of effective antipsychotic medications. Such activity is consistent with the possibility that viral infections may play a role in the pathogenesis of schizophrenia and other serious neuro-psychiatric diseases. This possibility is supported by our recent finding of particle-associated reverse transcriptase activity in the brains of some individuals with schizophrenia (Jones-Brando et al., 1995). The elucidation of the relationship between viruses, schizophrenia, and anti-viral medications may lead to new methods for the treatment of this disease and a new understanding of disease pathogenesis.

Acknowledgement

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: CEM-T4 cells from Dr. J.P. Jacobs, MT-2 cells, and AZT-resistant HIV-1 (A018) from Dr. Douglas Richman. The authors thank Dr. James Bremer at Rush-Presbyterian-St. Lukes Medical Center for providing fresh clinical isolates of HIV-1. This work was financed by the Theodore and Vada Stanley Foundation.

References

American Society of Health-System Pharmacists (1996) Clozapine. In: AHFS Drug Information 96. American Society of Health-System Pharmacists, Bethesda, MD. Pp. 1635-1646.

Amsterdam, J.D., Maislin, G. and Rybakowski, J. (1990a) A possible antiviral action of lithium carbonate in Herpes simplex virus infections. Biol. Psychiatry 27, 447-453.

Amsterdam, J.D., Maislin G., Potter, L. and Giuntoli, R. (1990b) Reduced rate of recurrent genital herpes infections with lithium carbonate. Psychopharmacol. Bull. 26, 343-347.

Ayuso, J.L. (1994) Use of psychotropic drugs in patients with HIV infection. Drugs 47, 599-610.

Baldessarini, R.J. and Frankenburg, F.R. (1991) Clozapine: a novel antipsychotic agent. N. Engl. J. Med. 324, 746-754.

Bohn, W., Rutter, G., Hohenberg, H. and Mannweiler, K. (1983) Inhibition of measles virus budding by phenothiazines. Virology 130, 44-55.

Breyer, U. and Villumsen, K. (1976) Measurement of plasma levels of tricyclic psychoactive drugs and their metabolites by UV reflectance photometry of thin layer chromatograms. Eur. J. Clin. Pharmacol. 9, 457-465.

Centorrino, F., Baldessarini, R.J., Kando, J., Frankenburg, F.R., Volpicelli, S.A., Puopolo, P.R. and Flood, J.G. (1994) Serum concentrations of clozapine and its major metabolites: effects of cotreatment with fluoxetine or valproate. Am. J. Psychiatry 151, 123-125.

Chang, T.-W. (1975) Suppression of herpetic recurrence by chlorpromazine. N. Engl. J. Med. 293, 153-154 (Letter).

Cheng, Y.F., Lundberg, T., Bondesson, U., Lindström, L., and Gabrielsson, J. (1988) Clinical pharmacokinetics of clozapine in chronic schizophrenic patients. Eur. J. Clin. Pharmacol. 34, 445-449.

Chou, J. and Chou, T.C. (1987) Dose-Effect Analysis with Microcomputers. Elsevier Science Publishers BV, Amsterdam.

Corbascio, A.N. and Bufalini, L.V. (1989) Possible neuroleptic-induced resistance to the human immunodeficiency virus. West. J. Med. 150, 92-93 (Letter).

Foley, G.E., Lazarus, H., Farber, S., Uzman, B.G., Boone, B.A. and McCarthy, R.E. (1965) Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18, 522-529.

Glatt, C.E., Snowman, A.M., Sibley, D.R. and Snyder, S.H. (1995) Clozapine: selective labeling of sites resembling 5HT₆ serotonin receptors may reflect psychoactive profile. Mol. Med. 4, 398-406.

Harada, S., Koyanagi, Y. and Yamamoto, N. (1985) Infection of HTLV-III/LAV in HTLV-1-carrying cells MT-2 and MT-4 and application in a plaque assay. Science 229, 563-566.

Hartvig, P., Eckernäs, S.A., Lundström, L., Ekblom, B., Bondesson, U., Lundqvist, H., Halldin, C., Någren, K. and Långström, B. (1986) Receptor binding of N-(methyl-¹¹C) clozapine in the brain of rhesus monkey studied by positron emission tomography (PET). Psychopharmacology 89, 248-252.

Jann, M.W., Grimsley, S.R., Gray, E.C. and Chang, W.-H. (1993) Pharmacokinetics and pharmacodynamics of clozapine. Clin. Pharmacokinet. 24, 161-176.

Jones-Brando, L., Ojeh, C., Herman, M.M., Kleinman, J.E., Hyde, T.M. and Yolken, R. (1995) Reverse transcriptase activity in human lymphocytes and brain tissue. Schizophr. Res. 15, 54-55 (Abstract).

Jones-Brando, L.V., Holland, L.E., Carbone, K.M., Yolken, R.H. and Torrey, E.F. (1996) Metabolites of clozapine inhibit the replication of neurotropic viruses. Schizophr. Res. 18, 143-144 (Abstract).

Kristiansen, J.E., Andersen, L.P., Vestergaard, B.F. and Hvidberg, E.F. (1991) Effect of selected neuroleptic agents and stereo-isomeric analogues on virus and eukaryotic cells. Pharmacol. Toxicol. 69, 299-403.

Larder, B.A., Darby, G. and Richman, D.D. (1989) HIV with reduced sensitivity to zodovudine (AZT) isolated during prolonged therapy. Science 243, 1731-1734.

Leysen, J.E., Janssen, P.M.F., Megens, A.A.H.P. and Schotte, A. (1994) Risperidone: a novel antipsychotic with balanced serotonin-dopamine antagonism, receptor occupancy profile and pharmacologic activity. J. Clin. Psychiatry 55(S), 5-17.

Libikowa, H., Stancek, D., Wiedermann, V., Hašto, J. and Breier, Š. (1977) Psychopharmaca and electroconvulsive therapy in relation to viral antibodies and interferon. Experimental and clinical study. Arch. Immunol. Ther. Exp. (Engl. Transl.) 25, 641-649.

Lieb, J. (1979) Remission of recurrent herpes infection during therapy with lithium. N. Engl. J. Med. 301, 942 (Letter).

Nemerow, G.R. and Cooper, N.R. (1984) Infection of B lymphocytes by a human herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists. Proc. Natl. Acad. Sci. USA 81, 4955-4959.

Nordström, A.-L., Farde, L., Nyberg, S., Karlsson, P., Halldin, C. and Sedvall, G. (1995) D₁, D₂, and 5-HT₂ receptor occupancy in relation to clozapine serum concentration: a PET study of schizophrenic patients. Am. J. Psychiatry 152, 1444-1449.

Patou, G., Crow, T.J. and Taylor, G.R. (1986) The effects of psychotropic drugs on synthesis of DNA and the infectivity of Herpes simplex virus. Biol. Psychiatry 21, 1221-1225.

Pauwels, R., Balzarini, J., Baba, M., Snoeck, R., Schols, D., Herdewijn, P., Desmyter, J. and De Clercq, E. (1988) Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J. Virol. Methods 20, 209-321.

Pearson, R.D., Manian, A.A., Harcus, J.L., Hall, D. and Hewlett, E.L. (1982) Lethal effect of phenothiazine neuroleptics on the pathogenic protozoan Leishmania donovani. Science 217, 369-371.

Perry, P.J., Miller, D.D., Arndt, SlV. And Cadoret, R.J. (1991) Clozapine and norclozapine plasma concentrations and clinical response of treatment-refractory schizophrenic patients. Am. J. Psychiatry 148, 231-235.

Schwartz, O., Henin, Y., Marechal, V. and Montagnier, L. (1988) A rapid and simple colorimetric test for the study of anti-HIV agents. AIDS Res. Human Retrovirus 4, 441-448.

Skinner, G.R.B. (1983) Lithium ointment for genital herpes. Lancet July 30, 288 (Letter).

Skinner, G.R.B., Hartley, C., Buchan, A., Harper, L. and Gallimore, P. (1980) The effect of lithium chloride on the replication of Herpes simplex virus. Med. Microbiol. Immunolo. 168, 139-148.

Torrey, E.F. (1988) Stalking the schizovirus. Schizophr. Bull. 14, 223-229.

Wunderlich, V. and Sydow, G. (1980) Lytic action of neurotropic drugs on retroviruses in vitro. Eur. J. Cancer 16, 1127-1132.

Wunderlich, V. and Zotter, St. (1982) Abrogation of infectivity of mouse mammary tumor virus by reserpine. Exp. Pathol. 21, 59-61.

Wunderlich, V., Fey, F. and Sydow, G. (1980) Antiviral effect of haloperidol on Rauscher murine leukemia virus. Arch. Geschwulstforsch. 50, 758-672.

Wyatt, R.G., Henter, I., Leary, M.C. and Taylor, E. (1995) An economic evaluation of schizophrenia – 1991. J. Soc. Psychiatry Psychiat. Epidemiol. 30, 196-205.

Ziaie, Z. and Kefalides, N.A. (1989) Lithium chloride restores host protein synthesis in Herpes simplex virus-infected endothelial cells. Biochem. Biophys. Res. Commun. 160, 1073-1078.