Original article
Opioid agonist – tachykinin antagonist as a new analgesic with adjuvant anticancer properties

Introduction

Opiate analgesics are widely used medicines to relieve severe acute or chronic pain. They are known to exert an antinociceptive effect by interaction with specific opioid receptors. However, the beneficial anal­gesic effect of opioids is accompanied by side effects such as constipation, vomiting and nausea. Tolerance and physical dependence on opioids are easily developed [9]. In a case of pains related to cancer, stimula­tion of cancer progression should be also considered [8]. In nociceptive signal induction, transmission and pain perception, different types of endogenous systems are involved. Therefore, development of new analgesics designed to treat cancer pain is an important target of medicinal chemistry [3]. New analgesics that may suppress pain signals in a different manner are re­cently developed as multitarget medicines, especially to treat chronic pain [6].

Tachykinins, including undecapeptide substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) play modulatory roles in the afferent transfer and postsynaptic processing of nociceptive information within the dorsal horn of the spinal cord [2,13]. The super­­ficial dorsal horn of the spinal cord is an area that receives primary synaptic input from sensory fibers originating from dorsal root ganglion (DRG) neurons. This area is a potentially important site for functional regulation (by modulatory opioid peptides) of nociceptive input mediated by substance P and excitatory amino acids released from primary afferent terminals. Ana­tomical studies indicate a similar distribution of substance P- and opioid-containing neural elements with­in this area [1,18]. Ligands of the opioid receptors are highly effective in blocking or reducing nociceptive transmission at the spinal level, and this antinociception is thought to be caused at least partially by inhibition of substance P release [5,13]. Therefore, simultaneous partial blocking of presynaptic substance P release and blocking of postsynaptic substance P receptors responsible for signal transmission has been the first multitarget approach. Indeed, co-injection of a weak peptide substance P antagonist together with an opioid peptide strongly enhances opioid analgesia [15]. Consequently, the development of multitarget ligands results in the development of a new type of effective analgesics [6]. Recently it has been published that opioid peptides [8] as well as substance P antagonists [7,16] express significant effects on progression of cancer cells. Therefore, potential reducing of cancer progression should be an additional advantageous effect of substance P-opioid peptide ligands use in the case of cancer-related pains.

Searching for a new type of analgesics we developed compound AA3016 (Fig. 1), analogue of previously synthesized AA501 [14], in which one opioid pharmacophore is hybridized with substance P antagonist mo­tive Z-D-Trp. This paper describes pharmacological binding as well as effect on proliferation of various cancer cells in vitro.

Material and methods

Compounds



Substance P and Lys-Phe-Phe-Gly-Leu-MetNH2 (AA2077) have been synthesized by solid phase synthesis using Fmoc-strategy, followed by preparative HPLC purification. AA3266 has been synthesized in solution methodology by an already patented method [10]. Aprepitant, 5-[[(2R, 3S)-2-[(1R)-1-[3,5-bis (trifluoro­methyl) phenyl] ethoxy]-3-(4-fluorophenyl)-4-mor­pho­linyl]methyl]-1,2-dihydro-3 H-1,2,4-triazol-3-one has been isolated from commercially available pills (Merck). The structure and purity of compounds were confirmed by HPLC-MS analysis. The compounds, opio­id antagonist naltrexone (NTX) and NK-1 antagonist – L703,606 have been purchased from Tocris.



Tachykinin hNK1 receptor binding



Neurokinin 1 receptor binding assays were perform­ed as previously described [19]. Briefly, recombinant hNK1/CHO cells were grown to 90% confluence in a humidified atmosphere (95% air and 5% CO2) at 37°C, in Ham’s F12 medium supplemented with 10% foetal bovine serum, 100 U/mL of penicillin, 100 g/mL of streptomycin and 500 g/mL of Geneticin (G 418). The cell monolayers were then washed with calcium and magnesium deficient phosphate-buffered saline (PD buffer) and harvested in the same buffer containing 0.025% EDTA. After centrifugation at 2700 rpm for 12 minutes, the cells were homogenized in ice cold 10 mM Tris-HCl and 1 mM EDTA (pH = 7.4) buffer. The crude membrane fraction was collected by centrifugation at 18,000 rpm for 12 minutes at 4°C, the pellet was suspended in 50 mM Tris-Mg buffer, and the protein concentration of the membrane preparation was determined using the Bradford assay. Six different concentrations of the test compound were each incubated, in duplicates, with 20 g of membrane homogenate and 0.5 nM [3H] Substance P (44.1 Ci/mmol, Perkin-Elmer, United States) in 1 mL final volume of assay buffer (50 mM Tris-HCl pH 7.4, containing 5 mM of Magnesium chloride, 50 g/mL of bacitracin, 30 M of bestatin, 10 M of captopril and 100 M of Phenylmethylsulfonylfluoride).

Substance P (10 M) was used to define the nonspecific binding. The samples were incubated in a shak­ing water bath at 25°C for 20 minutes. The reaction was terminated by rapid filtration through Whatman GF/B filter (Gaithersburg, MD) presoaked in 1% polyethyleneimine, washed three times with 2 mL of cold saline, and filter-bound radioactivity was determined by liquid scintillation counting (Beckman LS5000TD).

Data analysis was performed from three indepen­dent experiments by using the GraphPad Prizm4 soft­ware (GraphPad, San Diego, CA).

Log IC 50 values for each compound were determined from linear regression. The inhibition constant (Ki) was calculated using the Cheng-Prusoff equation [4].



Opioid MOR and DOR receptor binding



Opioid receptor binding assays were performed

as previously described [12]. The brain homogenates of Wistar rats (250-300 γ body weight) were used for further binding experiments. Animals were housed in the Mos­sakowski Medical Research Centre Polish Acade­my of Sciences, Warsaw, in groups of four, allowed free access to standard food and tap water, and maintained on a 12 : 12 h light/dark cycle until the time of sacrifice. Animals were handled according to the Directives of

the Council of European Communities (86/609/EEC) and the permission of the local Animal Ethical Commission.

All binding assays were performed at 25°C for 30 mi­nutes in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 ml, containing 1 mg of BSA and 0.2-0.4 mg/ml of membrane protein. The samples were prepared in disposable plastic assay tubes (Sarstedt Co., Nümbrecht, Germany). Rat brain membranes were incubated with the selective MOP receptor agonist [3H]DAMGO (0.9-1.2 nM) and the DOP receptor selective agonist ([3H]Tyr1,Ile5,6deltorphin-2; 0.8-1.3 nM) in the presence of unlabeled test ligands with concentrations ranging from 10-5 to 10-11 M. Non-specific binding was determined in the presence of 10 M naloxone. Three peptidase inhibitors (1 M captopril, 1 M bestatin and

1 M phosphoramidon) were included in the assay buffer to prevent metabolic inactivation of the peptides. The experiment was terminated and both bound and free radioligands were separated by rapid filtration under vacuum through Whatman GF/C (radiolabeled peptides) glass fibre filters by using Brandel M24R Cell Harvester. Subsequently, the filters were washed three times with 5 ml of ice-cold 50 mM Tris-HCl (pH 7.4) buffer. After completion of the filtration and separation procedure, all fibre-disks were dried under an infrared lamp and removed from the filter-sheet by use of tweezers. Each disk was inserted into UltimaGold™ environment friend­ly, non-volatile, toluene-free scintillation cocktail and placed into individual sample vials (transparent glass, Packard). The bound radioactivity was determined in a Packard Tricarb 2300TR liquid scintillation analyzer. Receptor binding experiments were performed in du­plicate and repeated at least three times.



[³⁵]GTPγS binding assay

Crude rat membrane preparation (10-15 g protein/

1 ml) was incubated for 60 min at 30°C in a Tris-EDTA (pH 7.4) buffer containing 50 mM of Tris-HCl, 1 mM of EDTA, 3 mM of MgCl2 and 100 M of GDP in the presence of 0.05 nM [³⁵]GTPγS and increasing concentrations (10-10– 10-5M) of the opioid-SP hybrid coded “3266” in a total volume of 1 ml. All experiments were performed in triplicate. Non-specific binding was determined by using 10 µM of unlabelled GTPS. The 3266 binding was reversed with an addition of either 10-5 M naloxone or a NK-1 antagonist – L703,606. Membra­ne-bound and free radioligand were separated by rapid triple filtration with Tris-EDTA (pH 7.4) buffer on Tris-EDTA-soaked glass microfiber GF/B Whatman filters using a M-24 Cell Harvester apparatus (Brandel, USA). Then, filter discs were soaked in an Ultima Gold MV scintillation fluid (Perkin Elmer, USA) and placed in glass tubes. Radioactivity was determined in a TRI-CARB 2100TR (Canberra-Packard, Perkin-Elmer Life Science, USA) scintillation counter. Binding curves were fitted using nonlinear regression and were depicted as a percentage of specific compound-stimulated [³⁵]GTPγS binding to a log of compound concentration. Efficacy was presented as the maximum possible effect (Emax) value.



Cell proliferation assays



Human melanoma cell lines MeW164, MeW155, MeW151, human lung cancer E14, urinary bladder carcinoma cell line T24, and also normal adult human fibroblast cell lines Fib9 and FlW180 and human foe­tal fibroblast cell line FlWp95 and FIW p98 were cultured in Eagle’s 1959 MEM (Biomed-Lublin) supplemented with 10% foetal calf serum (Invitrogen).

Adherent cell cultures were plated in 24-well plates and harvested at sub-confluency using 0.25% trypsin with EDTA (200 mg/L). The cells collected from the cultures were counted directly under a microscope. Cell viability was evaluated according to trypan blue staining and visualisation under an inverted phase-contrast microscope.

Results

Opioid receptor affinity of AA3266 in radioligand displacement binding assay



The radioligand displacement binding studies were performed with receptor-specific probes [3H]DAMGO and [3H]DELT II in rat membrane preparations. The opioid peptide codenamed AA3266 effectively displaced the binding of both specific radiolabeled agonists with an MOR IC50 0.06 nM ± 0.01 and DOR IC50 0.6 nM ± 0.24. The AA3266 peptide showed ten times higher affinity at the MOR opioid receptor than at DOR receptors.



3266-induced G-protein stimulation



The hybrid opioid-SP antagonist, AA3266 effectively stimulated G-protein activation in the rat membrane homogenate with efficacy characteristic of full agonists (Emax = 215.1 ± 5.7%) (Fig. 2). The extra sum of squares F test revealed that the addition of naloxone considerably decreased AA3266-induced G protein stimulation (F1,34 = 13.61; p < 0.001) but failed to abolish it fully (F1,34 = 27.54; p < 0.001), leaving the Emax value of 124 ± 3.4%. Whereas the NK-1 antagonist – L703,606 did not alter 3266-induced G-protein activity (F1,34 = 1.28; p = 0.26) as the Emax value equalled 220 ± 3.6% but in turn shifted the curve to the right affecting AA3266 potency (F1,34 = 5.91; p < 0.05). As a result, the potency of AA3266 was reduced by 0.3 log units (AA3266: logED50 = –6.7 ± 0.1; 3266 + L703,606: logED50 = –6.4 ± 0.06).

Receptor binding to tachykinin receptor hNK-1 is

Ki = 180.0 + 13.5 nM, that is similar to reference compound of C-terminal hexapeptide analogue (AA2077), Ki = 262.1 ± 14.3 nM [Tomczyszyn, in preparation].



,b>Effects of the compounds on the proliferation of normal and cancer cells



The compound AA3266 exerted a strong inhibitory effect on the proliferation of human melanoma cell lines (Fig. 3), and also inhibited proliferation of lung

cancer and urinary bladder cancer cells (Figs. 4 and 5).

The in­hibitory effect of AA3266 on human fibroblast lines was absent or found only at a dose of 100 M (Fig. 6). Aprepitant inhibited proliferation of human

cancer cell lines similarly as the compound AA3266 (Figs. 7-9). In contrast to AA3266, aprepitant efficiently inhibited proliferation of normal human fibroblast lines (Fig. 10). SP did not substantially inhibit proliferation of human normal or cancer cell lines (Figs. 11-14).

Discussion

Newly synthesized opioid agonist-substance P antagonist AA3266 expressed high affinity to both, MOR and DOR opioid receptors, with preference to MOR opioid receptors. The level of affinities is similar to biphalin that confirms the previous observation that one opioid pharmacophore with an additional lipophilic group on the other side of hydrazide bridge determines ‘biphalin like’ properties [11,12]. The affinity to human NK1 re­ceptors is similar to C-terminal hexapeptide fragment of substance P. Compound AA3266 in an in vitro cell breading test showed antiproliferative properties against several types of cancer cells. As previously described selective tachykinin antagonist aprepitant [17], the highest antiproliferative effect was exerted on melanoma cells. Interestingly, the antiproliferative properties against normal cells, i.e. fibroblasts were visibly lower.

To summarize, newly synthesized opioid agonist-tachykinin antagonist AA3266 expressed high affinity to opioid MOR and DOR receptors, and significant antagonist affinity to NK1 receptors. The compound expressed antiproliferative properties against various cancer cells. Therefore, the compounds applied in the treatment of chronic cancer-related pain may additionally enhance cancer directed therapies.

References

 1. Aicher SA, Punnoose A, Goldberg A. Mu-opioid receptors often colocalize with the substance P receptor (NK1) in the trigeminal dorsal horn. J Neurosci 2000; 20: 4345-4354.

 2. Carr DB, Cousins MJ. Spinal route of analgesia: Opioids and future options. In: Neural blocade in clinical anesthesia and management of pain. Cousins MJ, Bridenbaugh PO (eds.). 3rd ed. Lippincott Raven, Philadelphia 1998, pp. 915-983.

 3. Carr DB, Lipkowski AW. Today’s versus tomorrow’s opioids for cancer pain: who will close the gap? Analgesia 1996; 3: 227-235.

 4. Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099-3108.

 5. Jessel TM, Iversen LL. Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 1977; 268: 549-551.

 6. Kleczkowska P, Lipkowski AW, Tourwe D, Ballet S. Hybrid opioid/ non-opioid ligands in pain research. Curr Pharmaceut Design 2013; 19 [Epub ahead of print].

 7. Lazarczyk M, Matyja E, Lipkowski A. Substance P and its receptors – a potential target for novel medicines in malignant brain tumour therapies (mini-review). Folia Neuropathol 2007; 45: 99-107.

 8. Lazarczyk M, Matyja E, Lipkowski AW. A comparative study of morphine stimulation and biphalin inhibition of human glioblastoma T98G cell proliferation in vitro. Peptides 2010; 31: 1606-1612.

 9. Lipkowski AW, Carr DB. Rethinking opioid equivalence. Pain:

Clinical Updates 2002; 10/4: 1-4

10. Lipkowski AW. A method of producing novel opioid peptide. WO2010/077154.

11. Lipkowski AW, Misicka A, Davis P, Stropova D, Janders J, Lachwa M, Porreca F, Yamamura HI, Hruby VJ. Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin. Bioorg Med Chem Lett 1999; 9: 2763-2766.

12. Lukowiak M, Kosson P, Hennink WE, Lipkowski AW. The synthesis and pharmacological properties of new fluorescent opioid peptide analogue. Pharmacol Rep 2009; 61: 517-521.

13. Maszczynska I, Lipkowski AW, Carr DB, Kream RM. Dual functional interaction of substance P and opioids in nociceptive transmission: Review and reconciliation. Analgesia 1998; 3: 259-268.

14. Maszczynska Bonney I, Foran SE, Marchand JE, Lipkowski AW, Carr DB. Spinal antinociceptive effects of AA501, a novel chimeric peptide with opioid receptor agonist and tachykinin receptor antagonist moieties. Eur J Pharmacol 2004; 488: 91-99.

15. Misterek K, Maszczynska I, Dorociak A, Gumulka SW, Carr DB, Szyfelbein SK, Lipkowski AW. Spinal co-administration of peptide substance P antagonist increases antinociceptive effect of the opioid peptide biphalin. Life Sci 1994; 54: 939-944.

16. Munoz M, Rosso M, Covenas R. A new frontier in the treatment of cancer. NK-1 receptor antagonists. Curr Med Chem 2010; 17: 504-516.

17. Munoz M, Rosso M, Robles-Frias MJ, Salinas-MartIn MV, Rosso R, Gonzalez-Ortega A, Covenas R. The NK-1 receptor is expressed in human melanoma and is involved in the antitumor action

of the NK-1 receptor antagonist aprepitant on melanoma cell lines. Lab Investig 2010; 90: 1259-1269.

18. Regoli D, Boudon A, Fauchere J. Receptors and antagonist for substance P and related peptides. Pharmacol Rev 1994; 46: 551-599.

19. Yamamoto T, Nair P, Jacobsen NE, Vagner J, Kulkarni V, Davis P,

Ma SW, Navratilova E, Yamamura HI, Vanderah TW, Porreca F, Lai J, Hruby VJ. Improving metabolic stability by glycosylation: bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists. J Med Chem 2009; 52: 5164-5175.