A longevity study with enhancer substances ...

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Author's Personal Copy Life Sciences 182 (2017) 57–64

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A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain J. Knoll a,⁎, K. Baghy b, S. Eckhardt c, P. Ferdinandy a, M. Garami d, L.G. Harsing Jr a, P. Hauser d, Z. Mervai b, T. Pocza d, Z. Schaff e, D. Schuler d, I. Miklya a a

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine in Semmelweis University, Budapest, Hungary 1st Department of Pathology and Experimental Cancer Research, Faculty of Medicine in Semmelweis University, Budapest, Hungary National Institute of Oncology, Budapest, Hungary d 2nd Department of Pediatrics, Faculty of Medicine in Semmelweis University, Budapest, Hungary e 2nd Department of Pathology, Faculty of Medicine in Semmelweis University, Budapest, Hungary b c

a r t i c l e

i n f o

Article history: Received 20 December 2016 Received in revised form 9 June 2017 Accepted 12 June 2017 Available online 13 June 2017 Keywords: Enhancer-sensitive tumor-manifestation-suppressing (TMS) regulation Enhancer substances Selegiline/(−)-deprenyl (2R)-1-(1-benzofuran-2-yl)-N-propylpentane2-amine (BPAP) Fibromyxosarcoma

a b s t r a c t Aims: First proof to show that (−)-deprenyl/selegiline (DEP), the first selective inhibitor of MAO-B, later identified as the first β-phenylethylamine (PEA)-derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP), the tryptamine-derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer-sensitive brain regulations. Main methods: Longevity study disclosing the operation of tumor-manifestation-suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. Key findings: Whereas 20/40 saline-treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001 mg/kg DEP: 15/40 rats; with 0.1 mg/kg DEP: 11/40 rats (P b 0.01); with 0.0001 mg/kg BPAP: 8/40 rats (P b 0.001); with 0.05 mg/kg BPAP: 7/40 rats (P b 0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB-186 (Daoy); UW-228-2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine-transporter-2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi-modal, bell-shaped concentration-effect curves of DEP and BPAP. Significance: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMSregulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor. © 2017 Published by Elsevier Inc.

1. Introduction It was shown in earlier longevity studies performed on mammals with 0.25 mg/kg (−)-deprenyl/selegiline (DEP), which blocks selectively MAO-B activity in the brain, that DEP prolongs the life of rats [1–7], mice [8–9], Syrian hamsters [10], beagle dogs [11], and acts even on Drosophila melanogaster [12]. DEP was developed in the early 1960s as a psychic energizer [13]. The drug became known as the first selective inhibitor of B-type

⁎ Corresponding author at: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine in Semmelweis University, 1089 Nagyvarad ter 4., Budapest, Hungary. E-mail address: [email protected] (J. Knoll).

http://dx.doi.org/10.1016/j.lfs.2017.06.010 0024-3205/© 2017 Published by Elsevier Inc.

monoamine oxidase (MAO-B) [14]. A detailed study of DEP's peculiar catecholaminergic central stimulatory effect initiated the development of 1-phenyl-2-propylaminopentane (PPAP), a DEP analog without MAO-B inhibiting property acting similarly to DEP. This was positive proof that DEP's main central stimulatory effect unrelated to MAO-B inhibition [15]. DEP was finally identified as a catecholaminergic activity enhancer (CAE) substance, a synthetic analog of the natural CAE-substance β-phenylethylamine (PEA) [16]. Realizing that tryptamine is primarily a natural enhancer of the serotonergic neurons, initiated the development of (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2amine (BPAP), the tryptamine-derived, presently known, most potent synthetic enhancer substance [17]. The nature of the enhancer regulation; development of synthetic enhancer substances [18] and a review of the 50 year-history of DEP were recently published [19].

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J. Knoll et al. / Life Sciences 182 (2017) 57–64

A crucially important step forward in realizing physiological significance of the enhancer-regulation in the brain was the first longevity study performed with low enhancer doses of DEP and BPAP. The study revealed that the enhancer substances (DEP and BPAP) prolonged the life of rats in the low enhancer dose range and BPAP, the selective and much more potent enhancer substance than DEP, was the most effective in this respect. Thus, the longevity study adduced proof that the enhancer effect is responsible for the prolongation of life of mammals [20]. The first longevity study performed with the low, enhancer doses of DEP and BPAP gave us an astonishing, unforeseen surprise. The enhancer substances are highly specific markers of unknown enhancer sensitive brain regulations [21]. As presented in this paper, enhancer-treatment revealed the operation of a hitherto unknown enhancer-sensitive tumor-manifestation-suppressing (TMS) regulation in the rat brain. Around 50% of the Wistar rats used in our longevity studies spontaneously manifest a rapidly growing subcutaneous fibromyxosarcoma during their lifetime. We usually detect the appearance of the first tumor during the last quarter of their first year, and due to aging, more and more members of the group manifest the tumor. In our longevity study with BPAP, we found that treatment with 0.0001 mg/kg BPAP significantly decreased the manifestation of the malignant tumor. BPAP is the presently known specific, extremely potent, tryptamine-derived synthetic enhancer substance. “Enhancer regulation was originally defined as: the existence of enhancer-sensitive neurons capable of changing their excitability in a split second and working on a higher activity level, influenced by natural enhancer substances or their synthetic analogs” [21,p. 25]. Since BPAP, the best specific marker of enhancer-sensitive brain regulations, unexpectedly uncovered TMSregulation in rat brain, this is the first proof that the enhancer substances are specific markers of unknown enhancer-sensitive brain regulations.

2.2. Immunohistochemical identification of the fibromyxosarcoma in Wistar rats

2. Methods and materials

2.3. Cell culture studies

All experimental procedures were approved by local Ethical Committees and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals, 6th Edition, 2010.

2.3.1. Cell lines Human medulloblastoma cell line, HTB-186 (Daoy) was purchased from ATCC, and UW-228-2 was obtained via the courtesy of Professor Silber (University of Washington, Seattle, WA, USA).

2.1. Selection of the optimal doses of DEP and BPAP for the longevity study using the shuttle box technique

2.3.2. Maintenance HTB-186 (Daoy) and UW-228-2 cell lines were maintained in culture medium (each 500 ml Minimum Essential Medium Eagle, Alpha Modification (M8042, Sigma, St Louis, USA) with 50 ml FCS (Gibco), 40 mg Gentamycin (Sandoz), 5 ml sodium-pyruvate (S8636, Sigma, St Louis, USA), 5 ml non-essential-amino acid solution (M7145, Sigma, St Louis, USA), 10 ml L-glutamine (Sigma, St Louis, USA) at 37 °C in humified 5% CO2.

The acquisition of a two-way conditioned avoidance reflex (CAR) was analyzed during 5 consecutive days. The rat was put in a box divided inside into two parts by a barrier with a small gate in the middle, and the animal was trained to cross the barrier under the influence of a conditioned stimulus (CS, light flash). If it failed to respond within 5 s, it was trained with a foot-shock (1 mA), the unconditioned stimulus (US). If the rat failed to respond within 5 s to the US, it was classified as an escape failure (EF). One trial consisted of 10s inter-trial intervals, followed by 20s CS. The last 5 s of CS overlapped the 5 s US. At each learning session, the number of CARs, EFs and inter-signal reactions (IRs) were automatically counted and evaluated by multi-way ANOVA (for details see [20]). We performed the longevity study with 200 male Wistar rats, treating 10-week old rats until death. Groups of 40 rats were injected subcutaneously, three-times a week (Monday, Wednesday and Friday), with saline, DEP and BPAP, respectively. Group 1: Saline (0.9% NaCl) 0.05 ml/100 g; Group 2: DEP 0.1 mg/kg; Group 3: DEP 0.001 mg/kg; Group 4: BPAP 0.05 mg/kg; Group 5: BPAP 0.0001 mg/kg. DEP was supplied by Sanofi-Chinoin (Budapest, Hungary); BPAP by Fujimoto Pharmaceutical Company (Osaka, Japan); Tetrabenazine (synthetized by Prof. Csaba Szantay, Department of Organic Chemistry in the Technical University, Budapest, Hungary).

To prove the tumors' origin, immunohistochemically reactions were carried out on formalin-fixed, paraffin embedded sections. Following deparaffinization and rehydration, the slides were incubated by the following primary antibodies against vimentin (Dako, Glostrup, Denmark, 1:1200 dilution), smooth muscle antibodies (SMA, Dako, 1:400 dilution), desmin (Dako, 1:300 dilution), Ki67 (Dako, 1:100 dilution). The reactions were carried out in a Ventana Benchmarck XT automated immunohistochemical staining system (Ventana Medical System Inc., Tucson, AZ, USA) with HRP Multimer based, biotin-free detection method. Reagents and secondary antibodies were obtained from Ventana (iView DAB Detection Kit, Ventana). On several rats taken from each group, histological analysis was performed post mortem. The subcutaneous tumors were measured by the two largest diameters. The tumors were removed and fixed immediately after removal in 10% neutral formalin (in PBS, pH 7.0) for 24 h at room temperature, dehydrated and embedded in paraffin. 3–4 μm thick sections were cut and stained by hematoxylin and eosin (HE). The tumors were greyish-white and a soft consistency. Occasionally, hemorrhagic and necrotic areas of various degrees could be detected. Histologically, the tumor cells were round or elongated with round or oval nuclei and eosinophilic cytoplasm. Sometimes, mitotic figures were seen. The cells were embedded in a pale partly eosinophilic, partly basophilic loose matrix which contained areas of collagen fibers. The tumor infiltrated the subcutaneous tissues and the striated muscles. Immunohistochemistry proved the mesenchymal origin of the tumor cells, which strongly stained with vimentin; however reactions for SMA and desmin were negative. Ki67 was positive in up to 5% of the tumor cells, indicating the proliferation of tumor cells. The final histological diagnosis was fibromyxosarcoma in the subcutaneous tissue.

2.3.3. Proliferation assays In each well 3 × 103 HTB-186 or UW-228-2 cells were seeded in 96well plates (Sarstedt), solved in 100 μl of its own medium with 10% FCS. 24 h after seeding, cells were treated for 72 h by drugs solved in further 100 μl medium. First, both cell lines were treated by DEP and BPAP in monotherapy to determine its dose-effect curves in concentration of 10−6, 10−7, 10−8, 10−9, 10−10, 10−11, 10−12, 10−13 and 10−14 M. In combined treatment 10−3, 3.3 × 10−4, 1.1 × 10−4 and 3.7 × 10−5 M of temozolomide (Schering Plough, USA) or 0.04, 0.2, 1, and 5 μM of cisplatin (Ebewe Pharma, Austria) or 0,04, 0.2, 1, and 5 μM of etoposide (Ebewe Pharma, Austria) or 10−7, 10−6, 10−5 and 10−4 μM (UW2282) or 0.001, 0.005, 0.025 and 0.125 μM (HTB-186) of vincristine (Richter Gedeon, Hungary) were applied in monotherapy or combined with 10−13 or 10−8 M of DEP and BPAP. Cell proliferation was evaluated by MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay (M5655, Sigma) after a 72-hour treatment by the method described in manufacturer's protocol.

Author's Personal Copy J. Knoll et al. / Life Sciences 182 (2017) 57–64

2.4. Colon carcinoma liver metastasis model Colon 38 (C38) mouse colon carcinoma cell line maintained subcutaneously was utilized for the present study. C38 cells were isolated from subcutaneous tumor tissue by collagenase digestion (0.7 mg/ml) with subsequent filtration and washing steps. Afterwards, 30,000 tumor cells were inoculated into the spleen of each animal. Liver metastases appeared after 23 days. A total of 42 C57Bl/6 mice were used for the following experimental set-up. Group 1: Control; treatment: saline sc. from the following day after tumor cell inoculation (N = 10). Group 2: BPAP; treatment: daily 0.0001 mg/kg sc., from the following day after tumor cell inoculation (N = 8). Animals were terminated by cervical dislocation in ether anesthesia. Macroscopic metastases were counted, and liver samples were fixed in 10% formalin and embedded in paraffin for histological analysis. Statistical analyses were made by Graphpad Prism 4.03 software. Significance of change between control and BPAP-treated groups were assessed by using Mann-Whitney U test. Significance was declared at the standard p b 0.05 level. 2.5. BPAP reverses tetrabenazine induced decrease of [3H]dopamine release from rat striatum 2.5.1. Materials [3H]dopamine (dihydroxyphenylethylamine-3,4[3H], specific activity: 28.0 Ci/mmol), Soluene-350, and Ultima Gold XR liquid scintillation reagent were purchased from PerkinElmer Life and Analytical Sciences, Boston, MA, USA). BPAP ((−)1-(benzofuran-2-yl)-2propylaminopentane HCl) was received from Fujimoto Pharmaceutical Corp., Osaka, Japan [17]. Tetrabenazine was obtained from Sigma-Aldrich Kft, Budapest, Hungary. All other chemicals were of analytical grade. 2.5.2. Animals and drug treatments Male Wistar rats weighing 180–220 g were used for experiments. The animals were housed five to a cage in a temperature- and humidity-controlled animal facility on a 12-h light/dark cycle (6.00 a.m. on, 6.00 p.m. off) with food and water available ad libitum. Rats were treated with tetrabenazine (1 mg/kg sc.), BPAP (0.0001 g/kg sc.) or with their combination 60 min prior to decapitation and the release of [3H]dopamine from striatal slices was determined. Control rats were injected sc. with saline. 2.5.3. Preparation of rat brain slices. Rats were decapitated by guillotine, the brains were removed from the skull and the striatum was dissected and sliced as described by Glowinski and Iversen [22]. Striatal slices were collected and immersed in oxygenated (95% O2/5% CO2) Krebs-bicarbonate buffer (composition in mmol/L: NaCl 118, KCl 4.7, CaCl2 1.25, NaH2PO4 1.2, MgCl2 1.2, NaHCO3 25, glucose 11.5, ascorbic acid 0.3, and Na2EDTA 0.03) at room temperature. 2.5.4. Release of [3H]dopamine from rat brain slices. Rat striatal slices were loaded with [3H]dopamine (10 μCi) for 30 min in 1.5 ml aerated (95% O2/5% CO2, pH 7.4) and preheated (37 °C) Krebsbicarbonate buffer [23]. After loading the tissues with [3H]dopamine, striatal slices were transferred into low volume (0.3 ml) superfusion chambers (MSB GmbH, Heidelberg, Germany) and superfused with aerated and preheated Krebs-bicarbonate buffer. The flow rate was kept at 1 ml/min by a Gilson multichannel peristaltic pump (type M312, Villiers-Le Bel, France). The superfusate was discarded for the first 60 min period of the experiments, and then fifteen 3-min fractions were collected by a Gilson multichannel fraction collector (type FC203B, Middletown, WI, USA). To evoke stimulated [3H]dopamine efflux, biphasic electrical field stimuli (40 V voltage, 10 Hz frequency, 2-ms

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impulse duration for 3 min in fraction 4) were delivered by a Grass S88 Electrostimulator (Quincy, MA, USA). 2.5.5. Determination of [3H]dopamine efflux At the end of the superfusion, tissues were collected from the superfusion chambers, weighed and solubilized in 0.4 ml Soluene-350. An aliquot (50 μl) was mixed with 5 ml of liquid scintillation reagent and subjected to liquid scintillation spectrometry for determination of tissue content of radioactivity. The tissue content of [3H]dopamine was expressed as kBq/g tissue. To determine the radioactivity released from striatal slices, a sample (1 ml) of the superfusate was mixed with 5 ml of liquid scintillation reagent and subjected to liquid scintillation spectrometry. The efflux of [3H]dopamine was expressed as kBq/g/3 min fraction. To estimate the electrically induced [3H]dopamine overflow, the mean of the basal outflow determined before and after stimulation was subtracted from each sample and summed. The Quattro Pro and the GraphPad Prism computer programs were used for data calculation. 2.5.6. Statistical analyses The Student t-statistics for two-means and the one-way ANOVA followed by the Dunnett's test were used for statistical analysis of the data as appropriate. A level of probability (p) b 5% was considered significant. The mean ± S.D. was calculated and the number of independent determinations was indicated with n. 3. Results 3.1. The essence of the selection of the optimal doses of DEP and BPAP for the longevity study A bi-modal, bell-shaped concentration effect curve is characteristic to the enhancer substances. This peculiar behavior was noted in the course of our first experiments when we realized the CAE effect of DEP [24–25]. Nevertheless, only the exact analysis of the enhancer effect of BPAP, the selective and up to the present most potent enhancer substance, rendered the distinction of the ‘specific’ and ‘non-specific’ enhancer effect possible. The bi-polar, bell-shaped nature of the enhancer effect was confirmed on cultured rat hippocampal neurons [17]; and exactly analyzed on isolated locus coeruleus of rats. In this test, BPAP enhanced the activity of the noradrenergic neurons in the femto/picomolar concentration range with a peak at 10−13 (‘specific’ enhancer effect), and also in a 10 million times higher concentration range with a peak at 10−6 (‘non-specific’ enhancer effect) [26,27]. The in vivo effectiveness of DEP and BPAP was exactly analyzed earlier with the shuttle box technique [25]. For the longevity study, DEP's and BPAP's optimum doses with the ‘specific’ and ‘non-specific’ enhancer effect, respectively, were selected from the dose-effect curves published in reference [25]: f or DEP (Fig. 1) and BPAP (Fig. 2). The essence of the in vivo analysis was the measurement of the acquisition of a two-way conditioned avoidance reflex (CAR) in shuttle box and the inhibition of the rats' learning ability with tetrabenazinetreatment (1 mg/kg sc.) which reversibly blocks the vesicular monoamine transporter 2 (VMAT2). Tetrabenazine depletes at least 90% of norepinephrine and dopamine from their stores in the nerve terminals within 1 h. Due to the weak performance of the catecholaminergic brain engine, the activation of the cortical neurons remains below the level required for the acquisition of a CAR. According to our experience (we studied all notable drug families), tetrabenazine-induced inhibition of learning performance can only be antagonized by the administration of a synthetic CAE substance or by the complete inhibition of A-type MAO [15]. Selective inhibition of B-type MAO is ineffective. Besides DEP, Rasagiline is the only potent selective irreversible inhibitor of MAO-B in clinical use, and it is unable to antagonize tetrabenazine-induced inhibition of learning performance because it is devoid of the CAE effect [28].



Fig. 1. Suppression of tumor manifestation due to low dose treatment with enhancer substances. Treatment with saline versus DEP (A, B) and BPAP (C, D) in doses selected in the shuttle box test for the longevity study (see Methods and materials section). Statistics: Kaplan-Meier test, A: DEP 0.001 mg/kg P = 0.1155 (ns), B: DEP 0.1 mg/kg *P b 0.01 (P = 0.0054); C: BPAP 0.0001 mg/kg **P b 0.001 (P = 0.0005), D: BPAP 0.05 mg/kg **P b 0.001 (P = 0.0003).

3.2. Perception of an enhancer-sensitive TMS-regulation in the brain of Wistar rats From the beginning of the second year of treatment it seemed increasingly more likely that rats in the enhancer-treated groups would manifest the fibromyxosarcoma with a lower frequency as compared to the saline-treated group. The enhancer-treatment-induced highly significant suppression in the manifestation of the fibromyxosarcoma was first undeniable after 18-month treatment with 0.0001 mg/kg BPAP. Table 1 shows the individual differences in the time span of tumormanifestation in groups of 40 rats treated with saline or different doses of DEP and BPAP, respectively. In the saline-treated group of rats, the first tumor appeared in the 10th month of treatment by the end of the 18th month of treatment 14 rats had already manifested

Fig. 2. Enhancer-treatment-induced delayed tumor manifestation. One-way ANOVA followed by Neuman-Keuls post hoc test: Sal v. 0.0001 mg/kg BPAP **P b 0.01. Mean ± S.D.

the tumor. In striking contrast, none of rats treated with 0.0001 mg/kg BPAP manifested the tumor prior to the 19th month of treatment.

Table 1 Individual differences in the time span of tumor-manifestation in groups of rats, N = 40, treated with saline, or different doses of DEP and BPAP, respectively. Manifestation of tumors in the groups Age of rats (months)

Saline 0.5 ml/kg

10 11 12 13 14 15 16 17 18 19 20 21 23 24 25 26 27 28 29 30 31 32 Total number of tumors

1 2 2 2 1

DEP 0.1 mg/kg

DEP 0.001 mg/kg 1 1 2 2

BPAP 0.05 mg/kg

BPAP 0.0001 mg/kg

1

1 1 3 2 1 1 1 1

1 1 1 1 3 1 1

2

2 1 2

2 1 1 1 2

3

1

1

1

1

1 2 1

20

1 11

15

7

8


Thus, it was already clear in middle of the longevity study that an enhancer-sensitive TMS-regulation works in the rat brain. Fig. 1 shows that low dose treatment with enhancer substances suppress tumor manifestation. The percentage of tumors manifested in rats treated with 0.1 mg/kg DEP was already significantly (P b 0.01) lower than in saline-treated rats (Fig. 1B). Both in the lower, 0.0001 mg/kg dose (‘specific’ enhancer effect) (Fig. 1C) and in the higher, 0.05 mg/kg dose (‘non-specific’ enhancer effect) (Fig. 1D), BPAP was more effective than DEP in suppressing the manifestation of the fibromyxosarcoma. The data clearly demonstrates that the enhancer-sensitive TMS-regulation is decreasing with age in a similar way as the enhancer-sensitive catecholaminergic and serotonergic brain mechanisms, which work from weaning until sexual maturity, on a significantly higher activity level [29]. Sexual hormones (estrone, testosterone) then return the enhancer regulation to its pre-weaning level and the aging-related slow decay of the enhancer regulation continues until death [30]. The enhancer substances keep the TMS-regulation on a higher activity level. A lower number of rats manifested the tumor in their lifetime in the enhancer-treated groups (see Table 1), and as Fig. 2 shows the fibromyxosarcoma appeared later than in the saline-treated group. In comparison to saline-treatment, enhancer-treatment with 0.0001 mg/kg BPAP, the peak dose with the ‘specific’ enhancer effect, was the most efficient in this respect. Developing tumors in surviving enhancer-treated rats is proof that, due to aging, the decline of TMS-regulation arrived to a critical level and ceased to work. Accordingly, there is no difference in the microscopy and histology of the fibromyxosarcoma developed in saline-treated or enhancer-treated rats.

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3.5. BPAP reverses tetrabenazine-induced decrease of [3H]dopamine release in rat striatum The resting and the electrical stimulation-induced [3H]dopamine release measured from isolated striatal slices dissected from saline-treated rats are shown in Fig. 3A. The electrical stimulation-induced [3H]dopamine release was 15.19 ± 2.05 kBq/g in saline-treated rat striatum (n = 8). As shown in Fig. 3B, tetrabenazine pretreatment (1 mg/kg sc., 60 min prior to the experiment) significantly decreased the electrical stimulation-induced [3H]dopamine release from striatal slices of the rat ([3H]dopamine release: 6.51 ± 2.25 kBq/g, n = 8). The effect of 0.0001 mg/kg BPAP injected sc. 60 min before the experiment on [3H]dopamine release is shown in Fig. 3C. The electrical stimulation-induced [3H]dopamine release was 18.28 ± 0.58 kBq/g (n = 7) in striatal slices obtained from BPAP pretreated rats. The resting and the electrical stimulation-induced [3H]dopamine release measured from striatal slices of rats pretreated with both tetrabenazine (1 mg/kg sc.) and BPAP (0.0001 mg/kg sc.) is shown in Fig. 3D. The electrical stimulation-induced [3H]dopamine release was 16.89 ± 2.45 kBq/g (n = 7), which is significantly higher than the measurement from tetrabenazine-treated rat striatum. Fig. 4 and Table 2 summarize the electrical stimulation-induced [3H]dopamine release from striatum obtained from saline, tetrabe nazine, BPAP, and tetrabenazine plus BPAP-pretreated rats. As shown, the pretreatment of rats with tetrabenazine reduced the [3H]dopamine release evoked by electrical stimulation, and this reduced release was reversed by concomitant injections of tetrabe nazine and BPAP.

4. Discussion 3.3. Experiments on human cultured tumor cell lines proved that tumor cells are not enhancer-sensitive We tested the effect of BPAP and DEP on the proliferation of tumor cells in two types of human cultured medulloblastoma cell lines: Daoy HTB-186 originating from desmoplastic cerebellar medulloblastoma [31] and UW-228-2 originating from posterior fossa medulloblastoma with a diploid DNA content [32]. Considering the bi-modal, bell-shaped concentration effect curve characteristic to the enhancer effect of DEP and BPAP [24] we investigated the enhancers' effect within the 10−14 and 10−6 M range, in nine concentrations (see Methods and materials section). DEP and BPAP influenced in vitro the cultured tumor cells in a negligible degree and also in an opposite direction. The Daoy cell line was slightly inhibited by both DEP and BPAP; proliferation of UW-228-2 cells was slightly increased. Thus, BPAP and DEP are devoid of any notable direct cytotoxic effect on tumor cells. Moreover, the enhancer substances did not change the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine on the two human cultured medulloblastoma cell-lines (see details in Methods).

3.4. Colon carcinoma liver metastasis model BPAP inhibits in vivo metastasis formation of colon carcinoma cells in mice. We inoculated 30.000 mice colon carcinoma cells (C38) into the spleen of C57BI/6 mice. In the saline-treated group, the average number of macroscopic liver metastasis on Day 23 after inoculation was 14 tumors/liver. In a group of mice pretreated daily, subcutaneously, for one week with 0.0001 mg/kg BPAP prior to the tumor cell inoculation, and further treated until the end of the experiment (23rd day), only 2 tumors/liver appeared (p b 0.05). This finding clearly indicates that TMS-regulation also works in the mouse-brain.

Our present study was the first example showing how DEP and BPAP as markers detected the operation of the unknown enhancer-sensitive TMS-regulation in rat brain. Up to the present only the enhancer-sensitive catecholaminergic and serotonergic brain regulations were subject of detailed analysis and since TMS-regulation shows similar sensitivity toward DEP and BPAP and behaves in all aspects similarly to the catecholaminergic and serotonergic neurons, it is easy to interpret the findings presented in this paper. Due to the specific pharmacological spectrum of DEP, the only synthetic PEA-derivative devoid of the catecholamine-releasing property which enlightened that the catecholaminergic neurons belong to the enhancer-sensitive brain regulations, guided the study of this hitherto unknown life important mechanism in the mammalian brain. DEP is still registered in the text-books and used in research and therapy as the reference compound to block selectively MAO-B. Since the mid-1980s, however, deeper analysis of the characteristic enhancement of the catecholaminergic brain machinery in DEP-treated rats rendered probable that this effect is unrelated to the selective inhibition of MAO-B. The development of (−)-1-phenyl-2-propylaminopentane (PPAP), the DEP-analog devoid of MAO inhibitory property, being an equally active stimulant of the catecholaminergic neurons as DEP verified the suggestion [15]. The first study which demonstrated that multiple, low dose administration of DEP enhances catecholaminergic activity in the brain and this effect is unrelated to MAO-B inhibition allowed for the discovery of the enhancer sensitive brain regulations [25]. PEA and its best known synthetic derivatives (amphetamine and methamphetamine) are strong releasers of catecholamines from their plasmatic pools. Since the catecholamine releasing effect conceals the detectability of the enhancer-sensitive nature of the catecholaminergic neurons [18 Fig. 8], PEA's primer physiological function as a natural enhancer substance, as well as the fact that amphetamine and methamphetamine are, like DEP, PEA-derived synthetic enhancer substances, remained unknown. The later realization that tryptamine is like PEA a



Fig. 3. A. Resting and electrical stimulation (40 V, 10 Hz, 2-msec for 3 min)-induced [3H]dopamine release from rat striatal slices. Control experiments in which rats were treated with saline sc. 60 min prior to decapitation. Mean ± S.D., n = 8. B. Effect of tetrabenazine pretreatment (1 mg/kg sc. 60 min prior to decapitation) on resting and electrical stimulation-induced [3H]dopamine release from rat striatal slices. Note: tetrabenazine pretreatment reduced electrical stimulation-evoked [3H]dopamine release. Mean ± S.D., n = 8. C. Effect of BPAP pretreatment (0.0001 mg/kg sc. 60 min prior to decapitation) on resting and electrical stimulation-induced [3H]dopamine release from rat striatal slices. Mean ± S.D., n = 7. D. Effect of combined administration of tetrabenazine (1 mg/kg sc.) and BPAP (0.0001 mg/kg sc.) 60 min prior to decapitation on resting and electrical stimulation-induced [3H]dopamine release from rat striatal slices. Note: the electrical stimulation-evoked [3H]dopamine release was increased when tetrabenazine and BPAP were administrated concomitantly compared to tetrabenazine administration alone. Mean ± S.D., n = 7.

natural enhancer [24], signaled the elaboration of BPAP the most selective and potent synthetic enhancer substance currently known [17].

DEP is primarily a catecholaminergic enhancer (CAE) substance and is a weak enhancer of serotonergic neurons. BPAP, as a tryptamine-derivative, is a highly potent enhancer of serotonergic neurons, but it is even as a CAE substance much more potent than DEP. The catecholaminergic and serotonergic neurons were studied as the first models of the enhancer-sensitive brain regulations [21]. Regarding the mechanism of action of the enhancer-substances (see Figs. 3, 4 and Table 2), we have found that BPAP injected in a dose of 0.0001 mg/kg reversed the decrease in the electrical stimulation-induced [3H]dopamine release, evoked by 1 mg/kg of tetrabenazine in superfused rat striatal slices. Tetrabenazine is a VMAT2 inhibitor proposed to interact with extravesicularly located dihydrotetrabenazine Table 2 Electrical stimulation induced release of [3H]dopamine from striatal slices.

Fig. 4. Electrical stimulation-induced [3H]dopamine release from rat striatal slices obtained from saline, tetrabenazine (1 mg/kg sc.), BPAP (0.0001 mg/kg), and tetrabenazine (1 mg/kg) and BPAP (0.0001 mg/kg) pretreated rats. Rats were injected with drugs sc. Tetrabenazine pretreatment reduced [3H]dopamine release, (one-way ANOVA followed by the Dunnett's test, F(3,26) = 8.617, p = 0.0004, saline vs. tetrabenazine pretreated groups *p b 0.01) and this decrease in the [3H]dopamine release was reversed by BPAP administration (Student t-statistic for two means, t = 4.988, df = 13, #p b 0.0002). Mean ± S.D., n = 7–8.

Treatment

[3H]dopamine release (kBq/g)

1. Saline 2. Tetrabenazine 3. BPAP 4. Tetrabenazine and BPAP

15.19 ± 5.79 3.75 ± 4.18* 14.75 ± 6.58 15.49 ± 4.92#

Treatments: rats were injected with 1 mg/kg of tetrabenazine or with 0.0001 mg/kg of BPAP sc. Drugs were injected 60 min before the experiment. Rats were decapitated and striatal slices were prepared. The slices were loaded with [3H]dopamine and superfused. The resting and the electrical stimulation (40 V, 10 Hz, 2-msec for 3 min)-induced [3H]dopamine release was determined. One-way ANOVA followed by the Dunnett's test, F(3,26) = 8.617, p = 0.0004, saline vs. tetrabenazine pretreated groups *p b 0.0002. Student t-statistic for two means, 2:4 #p b 0.05. Mean ± S.D., n = 7–8.


binding site that is distinct from the dopamine uptake site on VMAT2. Moreover, tetrabenazine also binds to intra-vesicular dopamine release sites of VMAT2, exhibiting high and low sensitivity in binding affinity [33]. Both the extra- and intra-vesicular VMAT2 dopamine uptake and release sites may be involved in BPAP's effect. Furthermore, BPAP, acting as a substrate inhibitor of VMAT2, may compete with dopamine for uptake into the vesicle, and may exhibit a low affinity binding to the dopamine uptake site on VMAT2, as suggested by its poor activity on resting [3H]dopamine release in superfused striatal slices of the rat. The biphasic concentration-response curve for BPAP to release [3H]dopamine following electrical stimulation that fits a two-site model of interaction, supports the interaction with two different intra-vesicular sites, a high affinity (picomolar) site and a low affinity (μM) dopamine release site. A binding to the high affinity dopamine release site represents the specific catecholaminergic activity enhancer activity of BPAP [17], whereas the low affinity site is responsible for BPAP's non-specific enhancer effect of on [3H]dopamine release. The two dopamine release sites may be linked to two pools of dopamine within the vesicles, a free pool and a pool associated with the ATP complex [34]. Alternatively, these binding sites may regulate dopamine release from two distinct vesicular pools, the readily releasable dopamine stores and the long-term stores. It is also possible that the two binding sites evoke dopamine release with different mechanisms: the high affinity site mediates electrical stimulation induced release, whereas the low affinity site mediates the release of dopamine with reverse mode operation of VMAT2. Taking into account the dopamine uptake and release sites on VMAT2, which tetrabenazine and BPAP bind to, we concluded that the observed interaction of these two drugs in [3H]dopamine release may be related to a binding of BPAP to the high affinity intra-vesicular dopamine release site, which is, on the other hand, also sensitive to the tetrabenazine, the vesicular inhibitor. All in all, even the up to the present already verified details regarding the molecular mechanisms clarifying BPAP's highly characteristic bi-modal, bell-shaped concentration effect curves [26], testify that the discovery of the enhancer-sensitive brain regulations represents a promising new brain research domain. Prior to the discovery of the catecholaminergic activity enhancer effect of DEP and the discovery of the enhancer regulation in the mammalian brain, the hypothesis was defined in 1981 that a progressively developing catecholaminergic and trace-aminergic deficiency is responsible for the biochemical lesion in the aging brain which leads to the age-related decline in sexual and learning performance and ultimately natural death [35]. A further study proved that this effect of DEP is unrelated to the inhibition of MAO-B [29]. Decades ago, based on this concept, a retrospective analysis in parkinsonian patients was performed. The long term (9 years) effect of treatment with Madopar alone (N = 177) or in combination with Madopar + DEP (N = 564) revealed a significant increase in life expectancy in Madopar + DEP group regardless of the significant demographic differences between the two groups [36]. Enhancer substances keep the catecholaminergic neurons on a higher activity level. For example: 6.8 ± 0.18 nmol/g wet weight dopamine was released within 20 min from the substantia nigra isolated from saline treated rats and 14.8 ± 0.36 nmol/g wet weight dopamine was released within 20 min from the substantia nigra isolated from rats treated with a single dose of 0.0001 mg/kg BPAP [18 Table 10]. It is well known from studies with rodents and primates that dopaminergic neurons are silent or spontaneously active [37]. Treatment of rats with 0.0001 mg/kg BPAP obviously transforms the silent catecholaminergic neurons into spontaneous firing entities, thus, the discovery of the enhancer regulation explains the promptness of activation in assault/escape behavior [18 p. 12]. It is hard to overestimate the therapeutic consequences that 0.0001 mg/kg BPAP is capable to dramatically transform dopaminergic neuron's operation. Enhancer-sensitive regulations work in the uphill period of life, from weaning until sexual maturity, on a significantly higher activity level. Sexual hormones (estrone, testosterone) return the enhancer regulation to the pre-weaning level, putting into action the aging-related

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slow decay of the enhancer regulation which continues until death [29,30]. Synthetic enhancer substance slows the aging related decay of the enhancer sensitive brain regulations, improving the quality and duration of mammalian life. A recently published study illustrates the unique anti-aging effect of BPAP. We treated rats daily with saline versus 0.0001 mg/kg BPAP and measured in the shuttle box their ability to fix a conditioned avoidance reflex (CAR). Due to aging of the dopaminergic neurons, learning ability is subject of an age-related decline. We found in our recent study that the three month old group of saline-treated rats worked with full capacity and built on the 5th day of training in average nearly 90% of the possible 100% of CARs. Due to aging of the dopaminergic neurons, the group of 18-month-old saline-treated rats reached on the 5th day of training in average only b30% of the possible 100% of CARs. However, the group of 18-month-old rats treated daily with 0.0001 mg/kg BPAP produced on the 5th day of training in average N90% of the possible 100% of CARs [20 Fig. 7]. This is an unprecedented novelty. The discovery an enhancer sensitive TMS-regulation in a mammalian brain is further convincing evidence that regarding the enhancersensitive brain regulations we see the peak of an iceberg. 5. Conclusion DEP and BPAP, specific markers of hitherto unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMS-regulation in rat brain. The existence of a physiological malignant tumor-manifestation-suppressing regulation in a mammalian brain was never even considered. This enhancer-sensitive brain regulation works very likely also in other mammalian brains, including the human brain. To test in human the potential protective effect of BPAP seems highly reasonable because of the exceptional safety margin of this compound. BPAP exerts its specific enhancer effect in rats in 0.0001 mg/kg and even a 20,000-times higher dose is tolerated without significant toxic effect. Conflict of interest statement The authors declare no potential conflicts of interest.

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