Ultra-rapid brain uptake of subcutaneous sumatriptan in the rat: Implication for cluster headache treatment

Abstract

Background

In spite of the substantial therapeutic efficacy of triptans, their site of action is still debated. Subcutaneous sumatriptan is the most efficacious symptomatic treatment for cluster headache (CH) patients, showing therapeutic onset within a few minutes after injection even in migraine patients. However, whether subcutaneous sumatriptan is able to reach the CNS within this short time frame is currently unknown.

Methods

Here, by means of liquid chromatography/mass spectrometry, we investigated peripheral and brain distribution of subcutaneous sumatriptan soon after injection in rats at a dose equivalent to that used in patients. Tissue sumatriptan contents were compared to those of oxazepam, a prototypical lipophilic, neuroactive drug.

Results

We report that sumatriptan accumulated within brain regions of relevance to migraine and CH pathogenesis such as the hypothalamus and the brainstem as soon as 1 and 5 minutes after injection. Notably, sumatriptan brain distribution was faster than that of oxazepam, reaching concentrations exceeding its reported binding affinity for 5HT1_B/D receptors, and in the range of those able to inhibit neurotransmitter release in vivo.

Conclusion

Our findings indicate that sumatriptan distributes within the CNS soon after injection, and are in line with prompt pain relief by parenteral sumatriptan in CH patients.

Keywords

Triptans pharmacokinetics migraine

Introduction

Triptans currently represent the most specific symptomatic drugs for the treatment of migraine and cluster headache (CH) attacks. Several years after the approval of sumatriptan for migraine treatment, new derivatives had been developed to improve potency, efficacy as well as pharmacokinetic parameters (1,2). In spite of these therapeutic advancements, however, how and where triptans act to abort migraine and CH is still debated. Somehow mirroring the peripheral and central hypotheses of migraine pathogenesis (3 –5), it has been proposed that triptans act at both peripheral and central sites. A large body of preclinical evidence indicates that triptans counteract meningeal neurogenic inflammation via 5HT1_B receptor-dependent vasoconstriction, and/or 5HT1_D receptor-mediated reduction of proinflammatory peptide release from trigeminal terminals (6). However, more recent findings suggest that triptans act inside the CNS to inhibit trigeminovascular pain signaling. In particular, triptans accumulate within the intersynaptic space upon systemic injection and reduce neurotransmitter release in the rat (7). This is in keeping with the ability of sumatriptan to act presynaptically at central 5HT1_B/D receptors and reduce release of neuropeptides by central afferents of primary sensory neurons (8 –10). Indeed, intravenous sumatriptan blocks synaptic transmission between peripheral and central trigeminovascular neurons of rats (11). In keeping with this, about 10–20% of a sumatriptan dose accumulates in the cerebrospinal fluid upon i.v. injection (12).

On the clinical side, however, the poor ability of triptans to permeate the blood brain barrier (BBB) is still considered able to preclude a therapeutically-relevant effect within the CNS (13). In contrast with this interpretation, however, the well-known central side effects of triptans, such as drowsiness, sedation, nausea and dizziness, among others, suggest that these compounds permeate the BBB (14). Accordingly, a very recent study shows that subcutaneous sumatriptan competes with a radiotracer for binding to 5HT1_B receptors in multiple brain regions of migraine patients (15).

In spite of evidence that triptans distribute within the CNS (13), the question as to whether the temporal kinetics of triptan CNS distribution are consistent with the onset of therapeutic effects is still open. For instance, Deen and associates evaluated the impact of sumatriptan on radiotracer binding to brain 5HT1_B receptors 33–48 min after subcutaneous injection (15), even though this formulation prompts pain relief at earlier time points upon injection (16,17). Indeed, thanks to the very fast onset of effects, subcutaneous sumatriptan is the treatment of choice to quickly abort pain during CH attacks (18). In terms of onset of effects, according to clinical trials subcutaneous sumatriptan reduces CH pain and clinical disability within 5 min (18,19), and a specific time study (20) as well as clinical routine and anecdotal reports by CH patients indicate that complete pain relief can occur in a matter of seconds. Such an ultra-rapid effect suggests the ability of parenteral sumatriptan to readily reach the site of pain origin and/or the CH generator. In this regard, evidence that nausea occurs within 2–5 minutes after sumatriptan injection (21) indicate that the drug rapidly reaches the brain parenchyma. To our knowledge, however, there are no data on the ability of sumatriptan to enter the brain within this very short time frame. Such information might be of significance in better understanding sumatriptan pharmacokinetics, as well as helping to clarify how it may act to abort CH attacks and migraine. In the present study, we determined sumatriptan distribution in plasma, trigeminovascular and brain regions of relevance to craniofacial pain at 1 and 5 min after subcutaneous injection in the rat. To maintain consistency with the clinic, we adopted a dose/kg identical to that used in patients.

Methods

Animals

Adult male Wistar rats 230–250 g (Charles River, Milan, Italy) were maintained three per cage in a climate-controlled room at 23 ± 2℃ on a 12-hour light/dark cycles with free access to food and water. All animal manipulations were performed according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive 86/609/EEC). Randomisation was not conducted because of the experimental design of our study (single group of animals for the two time points). As for the power analysis, the experimental hypothesis of our study prompted us to consider the hypothalamic content of oxazepam as the parameter of reference. Unfortunately, we have been unable to find studies reporting the contents of oxazepam in this (or other) brain region of the rat. This precluded the possibility of defining an a priori hypothesis, as well as performing a sample size calculation. On this basis, to carry out our exploratory study, we chose a number of animals of 8 and 6 per group for the 1 and 5 min time points, respectively. Four rats were used for Evans Blue quantitation in the brain per each time point. This number was selected because of ethical reasons and also in light of the expectation that the variability of the tissue drug contents would not be high given the easily reproducible and standardised injection procedures.

Drug administration and animal perfusion

Rats received a subcutaneous injection (100 µl) of 0.086 µg of sumatriptan succinate in the right flank and an identical amount of oxazepam hydrochloride in the left flank. Both drugs were from Sigma-Aldrich (Milan, Italy). Animals were deeply anesthetised (1.5% isoflurane in 70% nitrous oxide and 30% oxygen) 30 sec and 4.30 min after the injection so that perfusion was started at 1 and 5 min after the injection. Transcardial perfusion was performed with cold saline by means of a peristaltic pump set at 3.5 ml/min. A blood sample of 50 µl was collected soon after the auricular incision and after 10 min the dura, trigeminal ganglion and brain rapidly collected and stored at −80℃. The multiple brain regions were dissected from frozen coronal sections (30 µm) obtained with a cryostat set at −20℃. Specimens were collected in Eppendorf tubes and stored at −80℃.

Evans blue quantitation

A 100 µl saline solution containing Evans blue (30 mg/kg, Sigma-Aldrich, MO, USA) was injected into rats via the tail vein and after 3 min the animals were transcardially perfused. At different time points, perfusion was stopped and a fraction of the cortex rapidly collected, weighted and homogenized for Evans blue quantitation. The tissue dye content was analysed by a blinded evaluator by means of HPLC and UV detection as reported (22).

Liquid chromatography/mass spectrometry analysis of sumatriptan and oxazepam

Upon a 10 min transcardial perfusion, the dura, trigeminal ganglion and the brain were rapidly collected. Brain regions were obtained from frozen coronal slices as reported above. Samples were weighed and diluted 1:5 (weight/volume) with a water/acetonitrile (30/70 %) solution containing 10 pg/µl of deuterated sumatriptan and oxazepam. Samples were homogenised by sonication, centrifuged at 25,000 RPM and supernatants collected and stored at −80℃. Sumatriptan and oxazepam, along with their respective deuterated internal standards, were quantitated by a blind evaluator in a 2 µl fraction of the samples using a liquid chromatography/mass spectrometry (LC/MS) apparatus (22). Liquid chromatography was conducted using a HPLC series 200 micro pump apparatus (PerkinElmer Life and Analytical Sciences) and a ZIC-HILIC column (50 × 2.1 mm i.d. 3.5-µm particle size 200 Å; Merck SeQuant; Umeå, Sweden). The LC-tandem mass spectrometry system consisted of a Perkin Elmer Sciex (Thornhill, ON, Canada) API 365 triple quadrupole mass spectrometer equipped with a TurboIonSpray interface.

Statistics

Data were analysed using the WinLTP 1.11 reanalysis program and the software package GRAPHPAD PRISM (version 4.0; GraphPad Software, San Diego, CA, USA). Numerical data are expressed as mean ± SD or ±SEM. Statistical significance was evaluated using paired two-tailed Student’s t-test or one-way ANOVA plus Tukey’s post hoc test. Differences were considered significant for p-values <0.05.

Results

Identification of a brain perfusion protocol

Exact quantitation of drug content in the brain extracellular space is biased by the contribution of drug present in the vascular bed. This is routinely circumvented by brain perfusion protocols, but whether perfusion parameters (i.e. flow and duration) are indeed sufficient to completely wash brain capillaries from blood is often not experimentally verified. In the present study, given that a possible partial brain perfusion would have significantly altered data interpretation, we first set up a perfusion protocol able to entirely wash the blood component from brain extracts. To this end, we took advantage of Evans blue, a dye that avidly binds to albumin and does not cross the BBB (23). The dye was injected i.v. and quantitated in the cortex of the perfused brains at different time points. As shown in Figure 1, we found that Evans blue concentrations decreased in the perfused cortex in a time-dependent manner, showing a first phase of rapid reduction and a delayed phase of slower decrease. Evans blue became undetectable after 10 min of perfusion, a time point we adopted as the one necessary to completely wash the brain capillary bed from injected drugs.

Figure 1.

Time course of Evans blue contents in brain extracts of rats upon different times of perfusion. The effects of different times of transcardial perfusion on the brain content of Evans blue is shown. A volume of 240 µl of a solution containing 25 mg/ml of Evans blue dissolved in saline was injected i.v. and the dye was measured in brain extracts at different times after perfusion (see Methods). Each point represents the mean ± SEM of four animals.

Comparison of sumatriptan and oxazepam brain uptake kinetics

Sumatriptan and oxazepam contents have been measured at 1 and 5 min after injection in the plasma, dura, trigeminal ganglion and multiple brain regions including the subfornical organ, the latter being a brain structure typically lacking the BBB. Both drugs were delivered subcutaneously at a dose/kg of 0.086 mg/kg equaling the human dose of sumatriptan (6 mg/70 kg). Oxazepam was selected as a reference compound because, beside being a well-known CNS permeant drug (24), it is also commercially available as a deuterated isotope, a key prerequisite for its reliable LC/MS quantitation (see Methods). Results are shown in histograms comparing tissue contents of the two drugs at the same time point (Figure 2(a) and (b)), or contents of a single drug at different time points (Figure 2(c) and (d)). As shown in Figure 2(a), we found that 1 min after injection, sumatriptan contents present in the dura mater, trigeminal ganglion and brainstem equaled those of plasma, whereas those found in the brain cortex, hypothalamus and subfornical organ significantly exceeded plasma levels (Figure 2(a)). To our surprise, at this very early time point the contents of oxazepam were similar all in the tissues analysed. Further, with the exception of those of the dura and trigeminal ganglion, the contents of oxazepam were lower than those reached by sumatriptan in plasma and corresponding CNS regions (Figure 2(a)). When drug exposure was increased from 1 to 5 min, the concentrations of sumatriptan increased in plasma, dura and brainstem, but did not change in the trigeminal ganglion, cortex, hypothalamus, and SFO (Figure 2(b) and (c)). As for the content of oxazepam at 5 min, we found that this did not change in the dura mater and trigeminal ganglion, whereas it significantly increased in plasma and in all the brain regions analysed (Figure 2(b) and (d)).

Figure 2.

Comparison of tissue distribution of sumatriptan and oxazepam in multiple peripheral and central tissues of rats. The contents of sumatriptan and oxazepam in plasma, dura, trigeminal ganglion (TG), cortex, hypothalamus, brainstem, and subfornical organ (SFO) are shown at 1 (a) and 5 (b) min after subcutaneous injection of 0.086 mg/kg. Tissue contents of sumatriptan (c) and oxazepam (d) at 1 and 5 min after injection. Each column represents the mean ± SD of eight (1 min) and six (5 min) animals per group. In (a) *p < 0.05, **p < 0.01 indicate statistically significant differences of sumatriptan tissue contents vs. plasma, and ^#p < 0.05, ^##p < 0.01 indicate statistically significant differences of oxazepam vs. sumatriptan contents in the respective tissue. In (b) *p < 0.05, **p < 0.01 indicate statistically significant differences of sumatriptan tissue contents vs. plasma, and ^#p < 0.05 indicates statistically significant differences of oxazepam tissue contents vs. plasma. In (c) and (d) ^§p < 0.05 and ^§§p < 0.01 vs. 1 min.

Discussion

The present study discloses a very rapid uptake of sumatriptan by different brain regions upon subcutaneous injection. Remarkably, we unexpectedly found that the temporal kinetics of sumatriptan brain distribution are faster than those of oxazepam, a prototypical brain permeant, neuroactive drug. Collectively, these findings are in keeping with recent evidence that sumatriptan displaces a radiotracer binding to brain 5HT1_B receptors (15), and corroborate prior work indicating the ability of sumatriptan to cross the BBB (see (13) for a comprehensive review). Data are also in line with the ability of subcutaneous sumatriptan to prompt quick migraine/CH relief, as well as central side effects soon after the injection (14,21). Clinical evidence demonstrates that functional disability improves in CH patients as soon as 0.5–5 min after subdermal administration (18 –20). It is conceivable, therefore, that such a very rapid relief from excruciating pain within seconds/minutes requires reaching the drug target with ultra-rapid temporal kinetics. This assumption is consistent with the present finding that sumatriptan is already accumulating in the rat brain 1 min after injection. We found that the hypothalamus is the brain region where sumatriptan accumulates more rapidly among the peripheral and CNS areas investigated. Of note, the hypothalamic contents equal those measured in the SFO, a periventricular organ that typically lacks the BBB. These findings therefore indicate that, at least in rodents, the hypothalamus has a high intrinsic permeability to sumatriptan, and, in light of the hypothalamic role in headache (25), point to this structure as a pharmacodynamically relevant site for the symptomatic effects of sumatriptan in CH patients. The brainstem showed sumatriptan contents analogous to those of the hypothalamus at 5 min after the injection, consistent with the hypothesis that the trigeminal nucleus caudalis is a site of action of sumatriptan (11). Even though our data do not allow claiming that sumatriptan exerts its pharmacotherapeutic effect by acting within the brain, they indicate that it is erroneous to exclude the possibility of a central action of sumatriptan merely on the basis of its supposed inability of cross the BBB. This appears to be of particular significance considering that the contrasting findings about the pathogenetic role of vasodilation in headache (26 –29) do not allow establishment of a causative role between triptan-dependent meningeal vasoconstriction and pain relief (30,31).

Apparently, the very fast distribution of sumatriptan within the rat brain is in contrast with its physicochemical properties. Among triptans, sumatriptan is considered the most hydrophilic, with an octanol/water partition coefficient (LogP) of 0.93, a value that is considered evidence of scarce CNS distribution. It should be noted, however, that morphine, a prototypical rapidly-acting neuroactive drug, actually shows an even lower LogP of 0.83. Likewise, the brain/plasma partition coefficient (K_p,brain) of sumatriptan is 0.13, a value almost identical to that of alfentanil (K_p,brain = 0.19), a very fast-acting and potent opioid (32). We reason, therefore, that the physicochemical properties of sumatriptan and its brain/plasma partition coefficient do not preclude, per se, either brain distribution or a central mode of action. Considering an approximate sumatriptan mean brain concentration of 100 pg/mg tissue (Figure 2(a)) and a rat brain weight of 1750 mg, the amount of sumatriptan reaching the brain within 1 min after injection is 1.03% of the dose. As for the mechanisms responsible for sumatriptan brain uptake, we reason that its passive permeability through the BBB and the inability of the P-glycoprotein to extrude it from the CNS (33), as well as the high brain blood flow, concur to its rapid brain distribution. Additional pharmacokinetic parameters such as plasma protein binding and cerebrospinal bulk flow may also play a role in regulating sumatriptan brain uptake. Further, the unexpected finding that at early time points after injection sumatriptan distributes within the CNS more promptly than oxazepam, a neuroactive and lipophilic (LogP = 2.2) drug, might be explained by considering that, at variance with sumatriptan, the high lipophilicity of oxazepam favors its initial interaction with the dermal adipose tissue. This might also cause a different contribution of the lymphatic system to sumatriptan and oxazepam systemic bioavailability. Indeed, small molecules preferentially enter the dermal capillaries rather than lymphatics (34), but a different interaction with dermal and subdermal tissue structures may inevitably affect absorption kinetics. Finally, it is worth noting that, as pointed out by Mahar Doan and colleagues (33), the permeability of the BBB to a given molecule cannot be merely predicted on the basis of its physicochemical properties because there is significant overlap in passive permeability, P-glycoprotein-mediated efflux and physicochemical properties between CNS penetrant and non-penetrant compounds.

The highest CNS contents we report here for sumatriptan are in the order of 150 pg/mg tissue that equal, upon a weight to volume conversion, a concentration of 510 nM. Remarkably, this value exceeds by about 30-fold the drug’s binding affinity (pKi) for 5HT1_B and 5HT1_D receptors and is similar to that able to reduce 5HT synaptic release in the cortex of freely moving rats (7). This suggests that sumatriptan reaches functionally relevant brain concentrations within 1 min upon injection, a scenario consistent with its very rapid activity in CH patients. Reportedly, sumatriptan alters serotonin and dopamine turnover 30 minutes post injection (35), but whether the drug exerts neurochemical effects at very early time points upon parenteral administration still waits to be determined. Our study, therefore, may promote investigations on early neuromodulatory effects prompted by sumatriptan, whose brain uptake was though, at best, to be minimal and delayed. Finally, it is worth noting that the concentrations of sumatriptan in the multiple cerebral regions are measured from extensively perfused rat brain, a procedure that might have dialysed part of the drug from the brain parenchyma, leading, therefore, to an underestimation of sumatriptan tissue contents.

In conclusion, the present study indicates that, upon subcutaneous injection at a dose equivalent to that used in patients, sumatriptan very rapidly reaches pharmacodynamically active concentrations within rat brain structures involved in headache pathogenesis. Data are in line with fast onset of pain relief in CH patients receiving sumatriptan injection, and with the hypothesis of a central mode of action of triptans.

Article highlights

Sumatriptan injected subdermally in rats at a dose equivalent to that adopted to treat cluster headache accumulates in brain regions of relevance to headache pathogenesis within 1 min.

The sumatriptan brain concentrations were higher than those of oxazepam, a neuroactive lipophilic drug, and exceeded the reported binding affinity for 5HT1_B/D receptors.

The inability of sumatriptan to cross the blood brain barrier and exert therapeutically-relevant central effects should be reconsidered.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from dxRare Disease Projects-Heath Projects 2007 and 2009, CR Foundation IG 2017 - ID. 20451 project.

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