Cancer Chemother Pharmacol (1987) 19: 131 - 132
`
`ancer
`hemotherapy and
`harmacology
`© Springer-Verlag 1987
`Chemotherapeutic agents do not interact
`with neurotransmitter receptors
`Stephen J. Peroutka
`Departments of Neurology and Pharmacology, Stanford University Medical Center, Stanford, CA 94305, USA
`Summary. The interactions of cisplatin, 5-fluorouracil,
`doxorubicin, mitomycin, carmustine (BCNU), cyclophos-
`phamide, methotrexate and thio-TEPA were assessed at
`three neurotransmitter receptor binding sites. Each drug
`was inactive at concentrations as high as 10-4M in dis-
`placing the specific binding of 3H-spiperone to dopamine
`Dz, 3H-pyrilamine to histamine H1, and 3H-quinuclidinyl
`benzilate to muscarinic cholinergic receptors. These data
`suggest that chemotherapy-induced nausea and vomiting
`are not due to interactions with neurotransmitter recep-
`tors.
`Introduction
`Nausea and vomiting secondary to chemotherapeutic
`agents are a major cause of morbidity in cancer therapy
`[1]. Gastrointestinal upset occurs in most patients treated
`with drugs such as cisplatin and may be the most debilitat-
`ing side-effect of the therapy. Nausea and vomiting may
`actually limit the course of chemotherapy in many patients
`[3]. However, the mechanism of action by which chemo-
`therapeutic agents produce these side-effects is unknown.
`While it has been postulated that drugs such as 5-fluorou-
`racil act directly on the chemoreceptor trigger zone in the
`central nervous system, drugs such as cisplatin are be-
`lieved to induce emesis via peripheral effects [3].
`By contrast, the efficacy of many antiemetic agents ap-
`pears to derive from their ability to block dopamine D2,
`histamine HI, and/or muscarinic cholinergic receptors [2].
`Moreover, these neurotransmitter receptors have been
`identified in brainstem pathways (e.g., area postrema) that
`are believed to mediate nausea and vomiting [2]. In addi-
`tion, these neurotransmitter receptors are also present out-
`side the central nervous system [4]. Conceivably, chemo-
`therapeutic agents may also interact with the same neuro-
`transmitter receptor sites as are blocked by classical an-
`tiemetics. Therefore, the present study was conducted to
`evaluate whether several commonly used chemotherapeu-
`tic agents could compete for radioligand binding to specif-
`ic neurotransmitter receptor sites.
`* This work was supported in upart by the John A. Hartford
`Foundation and NASA grant NCA 2-1 R 745-504
`Offprint requests to: S. J. Peroutka
`Materials and methods
`Radioligand studies were performed in brain membranes
`as previously described [2]. Adult rat brains were obtained
`from Pel-Freez Biologicals (Rogers, Ark) and stored at
`- 20 ° C until needed. On the day of study, the brains were
`defrosted, and the cortex and caudate were dissected. Tis-
`sues were homogenized in 20 vol. 50 mM Tris-HC1 (pH
`7.7 at 25 ° C) using a Brinkmann Polytron and then centri-
`fuged in an IEC B20A centrifuge at 49000 g for 10 min.
`The supernatant was discarded, and the pellet was resus-
`pended in the same volume of Tris-HC1 buffer and incu-
`bated at 37 °C for 10 min prior to a second 10-min centri-
`fugation at 49 000 g. The final pellet was resuspended in 80
`vol. Tris-HC1 buffer containing 10 ~tM pargyline, 4 mM
`calcium chloride, and 0.1% ascorbic acid. The suspensions
`were immediately used in the binding assay.
`Binding assays for drug displacement studies consisted
`of 0.1 ml 3H-ligand [final concentrations: 0.6-0.8 nM 3H-
`spiperone; 1.5-2.0 nM 3H-pyrilamine; 0.3-0.4 nM 3H-
`quinuclidinyl benzilate (QNB)], 0.1 ml buffer of displacing
`drug and 0.8 ml tissue suspension. Following incubation at
`25 °C for 30 min, the assays were rapidly filtered under
`vacuum through Whatman GF/B filters with two 5-ml
`washes using 50 mM Tris-HC1 buffer. Radioactivity was
`measured by liquid scintillation spectroscopy in 5 ml
`Aquasol (New England Nuclear; Boston, Mass) at 54% ef-
`ficiency. Specific binding was defined using 1 p~M (+)but-
`aclamol for 3H-spiperone binding in caudate membranes,
`1 gM chlorpheniramine for 3H-pyrilamine binding, and
`1 gM scopolamine for 3H-QNB binding (both performed
`in cortical membranes). Generally, 75%-90% of total bind-
`ing was specific for each radioligand. Radioligands were
`obtained from Dupont-New England Nuclear (Boston,
`Mass), and drugs were obtained from commercial sources.
`Results
`The interactions of cisplatin, 5-fluorouracil, doxorubicin,
`mitomycin, BCNU, cyclophosphamide, methotrexate, and
`thio-TEPA were assessed at three neurotransmitter recep-
`tor binding sites. The ability of each drug to compete for
`the specific binding at each neurotransmitter receptor site
`was analyzed using drug concentrations between 10 -6 and
`10 -4 M. Each experiment was performed in triplicate and
`repeated three times. None of the eight agents significantly
`inhibited the specific binding of 3H-spiperone, 3H-pyril-
`HELSINN EXHIBIT 2084
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00948
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`132
`amine, or 3H-QNB. Therefore, the eight chemotherapeutic
`agents did not interact with dopamine D2, histamine H1 or
`muscarinic cholinergic receptors at concentrations as high
`as 10 -4 M.
`Discussion
`The major finding of the present study is that commonly
`used chemotherapeutic agents do not bind to neurotrans-
`mitter receptor binding sites that are believed to be in-
`volved in the pathogenesis of nausea and vomiting [2]. The
`ability of many antiemetics to block dopamine D2, hista-
`mine H 1 and/or muscarinic cholinergic receptor sites
`strongly suggest that these receptors are involved in the
`mediation of nausea and vomiting [3]. The ability of
`chemotherapeutic agents to induce nausea and vomiting
`must therefore result from either a direct (primary) or an
`indirect (secondary) effect on central emetic pathways.
`Since the mechanism by which chemotherapeutic agents
`cause nausea and vomiting is unknown, direct activation
`of neurotransmitter receptors must be considered a poten-
`tial action of these drugs. However, the inability of the
`eight agents to interact with dopamine D2, histamine H l
`and muscarinic cholinergic receptors suggests that activa-
`tion of central emetic pathways is a secondary rather than
`a primary effect of cancer chemotherapeutic agents.
`Acknowledgements. I thank Faith H. Smith for assistance in manu-
`script preparation.
`References
`1. Laszlo J, Lucas VS (1981) Emesis as a critical problem in
`chemotherapy. N Engl J Med 305:948
`2. Peroutka S J, Synder SH (1982) Antiemetics: Neurotransmitter
`receptor binding predicts therapeutic actions. Lancet 1:
`658
`3. Seigel LJ, Longo DL (1981) The control of chemotherapy-in-
`duced emesis. Ann Intern Med 95:352
`4. Snyder SH (1983) Neurotransmitter receptor binding and
`drug discovery. J Med Chem 26:1667
`Received July 8, 1986/Accepted September 10, 1986
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