INTRANASAL ADMINISTRATION FOR A SUSTAINED BRAIN DELIVERY OF HIGHLY PROTEIN-BOUND DRUGS

Technical Field

[0001] The present disclosure relates to the field of medicine, more specifically to the field of chronic central nervous system disorders, including depression.

[0002] The present invention discloses a method for the controlled release of highly plasma proteinbound drugs to the brain by means of intranasal administration, demonstrating that the intranasal route sustains highly plasma protein-bound drug release to the brain, in comparison with intravenous (IV) and oral routes, reducing lung drug exposure and consequently envisioning the reduction of pulmonary side effects.

Background

[0003] Central nervous system (CNS) disorders comprise psychiatric (e.g. depression), neurological diseases (e.g. epilepsy) and neurodegenerative (e.g. Alzheimer's disease and Parkinson's disease). Neuroinflammation underlies their physiopathology. Most of those diseases currently have no cure, although symptomatic treatments are clinically available. Oral administration is the most practical and preferable route for the chronic treatment of CNS disorders, while intravenous (IV) injection is chosen for acute and emergency complications, such as status epilepticus. Both routes require drug passage across the blood-brain barrier (BBB) to reach the injured brain tissue and provide therapeutic effects. Nonetheless, centrally acting drugs (e.g. antidepressants, antipsychotics, antiepileptic drugs) are difficult to titrate and imply therapeutic drug monitoring. In addition, they can cause dose-dependent side effects, and are associated with high inter- and intra-individual variability particularly regarding their hepatic metabolism [1-8].

[0004] Intranasal administration of centrally acting drugs is of interest due to its clinical advantages: improvement of drug bioavailability by direct systemic absorption through the respiratory mucosa and avoidance of intestinal and hepatic first-pass metabolism; direct nose-to-brain drug delivery; and reduction of drug-drug interactions (DDIs) during intestinal absorption and hepatic metabolism [9- 12]. Furthermore, its ease-of-use, the possibility of self-administration and non-invasiveness also improve patient compliance. The increment of brain levels of small and lipophilic drugs comparatively to IV administration has also been demonstrated [13-15]. However, despite being very permeable across biological membranes, lipophilic compounds may also strongly bind to plasma proteins. Since only the free fraction is able to cross the BBB and interact with therapeutic targets, changes in plasma protein binding may interfere with drug disposition and, consequently, pharmacological effects. This is clinically relevant for compounds that are more than 90% bound to plasma proteins. Moreover, particularly at higher doses, saturation of binding sites of plasma proteins, as well as of transporters and metabolic enzymes, are likely to occur and contribute to variations of drug plasma concentrations and effects. Nothing has yet been reported regarding plasma protein binding and intranasal administration.

[0005] By providing direct nose-to-brain transport and offering enhanced targeting ability, as well as reduced systemic side effects, the intranasal route has been highlighted as a potential strategy for drug delivery to the brain [16].

[0006] Nevertheless, this pathway is not indiscriminately applicable to all chemical entities. In fact, low-solubility, low permeant and/or less potent drugs require additional efforts to develop formulations, such as nanosystems and triggered formulations, which confer protection from chemical/metabolic degradation, enhance drug solubility and prolong nasal residence time. On the other hand, small molecules such as the antiepileptic drugs levetiracetam and zonisamide [13, 14], and lipophilic compounds such as quetiapine and risperidone [15, 17], seem to undergo direct nose- to-brain delivery and exhibit decreased systemic exposure.

[0007] For instance, the pharmacokinetics of levetiracetam after intranasal administration of 625 pg per mice resulted in a bioavailability of approximately 107.44 %, with rapid and extensive systemic absorption [14]. The similar required time to reach maximum concentration (tmax) after intranasal and IV administrations (5 min), as well as the higher concentrations found in brain after intranasal instillation, suggest the advantageous use of this route for the delivery of levetiracetam in emergency situations, bypassing the invasive IV technique and allowing self-administration by patients or caregivers. Indeed, the brain exposure of levetiracetam after intranasal administration almost doubled comparatively to IV injection (742.41 vs 381.93 pg.min/mL) and drug targeting efficiency (DTE) was 182.35 %. These findings evidence the direct nose-to-brain delivery of levetiracetam and create a new hope for the treatment of refractory epilepsy.

[0008] In opposition, when administered by intranasal instillation, zonisamide was not completely absorbed (bioavailability of 54.95 %), but quickly attained the systemic bloodstream (tmax = 5 min) [13]. In brain tissue, the concentrations achieved at 5 min post-intranasal administration were 8 times higher than those achieved by IV administration. The DTE of 149.54 % and the direct transport percentage (DTP) of 33.13 % undoubtedly emphasize direct nose-to-brain delivery, the latter meaning that more than one third of the drug reached the brain through direct pathways. In contrast, renal exposure after intranasal administration was considerably reduced in relation to IV or oral routes. This fact is of utmost importance, because it decreases the likelihood of renal lithiasis, which is the adverse effect most often associated with zonisamide.

[0009] Both levetiracetam and zonisamide bind negligibly to plasma proteins (< 10% and 40%, respectively) [18, 19]. The results are promising particularly for emergent clinical situations such as seizures and status epilepticus since tmax and Cmax in the brain are comparable between intranasal and IV routes.

[0010] Affecting more than 300 million people and representing 4.4% of disease burden, depression is the mood disorder with the highest incidence worldwide. The World Health Organization reported that depression is the leading cause of disability and the fourth leading cause of premature mortality, generating substantial costs to health systems and a dramatic reduction of patient's quality of life [20]. With the Covid-19 pandemic, these numbers increased even more [21] and the prevalence of depression is expected to increase exponentially in the upcoming years. Despite the wide variety of pharmacological treatments available in clinical practice, the success rate of antidepressant drugs is only around 60-70% and there is a considerable inter-individual variability of drug response [22]. Reduction of variability and novel therapeutic alternatives remain an unmet clinical need.

[0011] Sertraline hydrochloride, also known as (lS-cis)-4-(3,4-dichlorophenyl)-l,2,3,4-tetrahydro- N-methyl-l-naphthalenamine hydrochloride (Figure 1), is a selective serotonin reuptake inhibitor (SSRI) clinically used as antidepressant.

[0012] The Food and Drug Administration (FDA) has also approved sertraline for the treatment of obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, premenstrual dysphoric disorder, and social anxiety disorder [23].

[0013] Sertraline's antidepressant action results from its primarily inhibitory effect on presynaptic serotonin reuptake, leading to the accumulation of serotonin in the synaptic cleft and regulation of the mood, personality, and wakefulness of depressed patients.

[0014] A major drawback of depression treatment with oral SSRIs (including sertraline) is the lag time period between the beginning of the treatment and the onset of action, which is, at least, two weeks. This delay has been explained by several factors namely the activation of serotonin receptors, particularly serotonin receptor 1A (5-HT1A). The increase of synaptic serotonin levels stimulates 5- HT1A receptors that are part of a feedback inhibitory system, lowering the release of serotonin from pre-synaptic neurons. Over the weeks, 5-HT1A receptors are desensitized, the feedback inhibitory system is inactivated and serotonin release is normalised which, together with the inhibition of serotonin reuptake by sertraline, increase serotonin concentrations in the synaptic cleft [24]. It becomes hence evident that the delayed response of SSRIs, including sertraline, is related to pharmacodynamic mechanisms with sustained drug levels. It does not depend on how fast it attains the brain. Indeed, past controlled studies have failed to show a faster onset of action for SSRIs administered by IV route compared to oral administration [25-27].

[0015] This corroborates that achieving stable sertraline concentrations in the brain, in steady-state and during a prolonged period of time, is clinically more relevant than reaching quick and high sertraline concentrations.

[0016] Importantly, for antidepressant drugs that require prolonged treatments and that exhibit a similar lag time between IV and oral routes, early achievement of high peak concentrations in the brain is not mandatory. In fact, a sustained drug delivery to the brain is advisable to avoid peak values that hamper drug titration and increase response variability. For this reason, in clinical practice, treatment with antidepressants normally continues after a successful resolution of symptoms has been achieved, in order to minimize the risk of relapse.

[0017] Oral formulations are currently the only marketed form of SSRIs, including sertraline. Although its use is relatively safe, certain adverse effects have been described, namely drug-induced lung diseases such as interstitial lung disease and bronchiectasis [28-30]. The analysis of postmortem fluids and tissues of 11 aviation accident victims demonstrated that the distribution coefficient of sertraline after oral administration is extremely high in lungs (mean value of lung/blood ratio is 67), which can justify its lung-toxicity [31]. Lung disease can progress rapidly, and lead to respiratory failure and death if medication is continued.

[0018] After oral administration, sertraline is quickly absorbed (tmax = 4-8 h) but its bioavailability is only around 44%, mainly due to its significant first-pass metabolism mediated by the isoforms of cytochrome P450 (CYP), CYP3A4, CYP2C9 and CYP2D6. Most of these are associated with genetic polymorphisms that contribute to inter-individual variability [32].

[0019] Sertraline absorption is slightly increased (25% higher AUC) and accelerated (tmax = 2.5h) when orally administered with food, which aggravates response variability due to drug-food interactions. Moreover, as previously referred, sertraline is highly bound to plasma proteins (98%), exhibiting a significantly smaller mean percentage of unbound drug in the elderly (1.42%) than in young individuals (1.55%; p < 0.001). Smaller unbound drug values are also reported in females (1.44%) than in males (1.52%; p < 0.012) [32],

[0020] All the aforementioned characteristics underlying pharmacokinetic variability make the oral treatment with sertraline quite challenging and hamper its titration. Furthermore, its increased potential to develop peripheral side effects and DDIs may lead to subtherapeutic or toxic effects.

[0021] On the other hand, neuroinflammation had been demonstrated to underly the physiopathology of several CNS-diseases, including Parkinson's disease [33], Alzheimer's disease [34], dementia [35], cerebral tumours [36] and depression [37]. Therefore, the use of anti- inflammatory drugs, including non-steroid anti-inflammatory drugs, have been demonstrating to have a high potential to reduce neuroinflammation [38]. However, this implies a chronic administration that can be impaired by the development of severe systemic side effects. In this way, intranasal administration is expected to reduce drug toxicity and improve brain sustained delivery. Therefore, herein, piroxicam was selected to be administered by intranasal route, particularly because it binds 99% to plasma proteins [39], and is classified as a Class II drug regarding the Biopharmaceutics Classification System.

[0022] In the last decade, the intranasal route has received increasing attention for the administration of CNS-acting drugs by enabling direct nose-to-brain delivery besides systemic drug absorption [12, 40, 41]. Most drug delivery systems encompass nanoparticle-based formulations, in particular nanoparticles, (nano)suspensions, (nano)emulsions, whereas aqueous solutions or thermoreversible gels are scarce. Options involving the use of triggerable systems, in particular those that are thermoresponsive such as in situ gelling formulations, have also been explored for intranasal drug delivery. Their greater residence time in the nasal mucosa due to higher viscosity leads to better performance, when compared with the corresponding solutions. An example in which this strategy was successfully employed involves the antidepressant venlafaxine, which was loaded into thermoresponsive polymer Lutrol® F127 and an array of mucoadhesive polymers (Carbopol® 934P, HPMC K4M, PVP K30, sodium alginate, tamarind seed gum and carrageenan) [42].

[0023] However, due to the small volume of nasal cavity and mucociliary clearance, intranasal administration is usually used to administrate potent drugs and nasal formulations shall be in a preferred embodiment mucoadhesive to retain drugs for prolonged periods.

[0024] Currently, there are no intranasal formulations of sertraline on the market.

[0025] These facts are introduced to substantiate the scientific problem addressed by the present disclosure.

General Description

[0026] Plasma protein binding refers to the degree to which a drug binds to plasma proteins. Drug efficacy is affected by the degree to which it binds to plasma proteins. Less protein-bound drugs can cross cell membranes and interact with pharmacological targets more efficiently.

[0027] The present invention is based on the surprising and unexpected identification that the intranasal administration of drugs that bind highly to plasma proteins, such as sertraline and piroxicam, increases their systemic bioavailability and sustains their distribution to the brain and peripheral tissues. [0028] For purposes of interpretation of the present invention, the expression "drug highly binding to plasma proteins" shall be interpreted as drugs having at least 90% of binding to plasma proteins, preferably at least 95% of binding, most preferably at least 98% of binding and even more preferably 99% of binding to plasma proteins.

[0029] In an embodiment, the present invention covers highly plasma protein-bound drugs which are active in the central nervous system.

[0030] In a specific embodiment of the present invention, the antidepressant drug sertraline, which binds highly to plasma proteins with an extent of 98% [20], surprisingly exhibited a more sustained and controlled distribution into the brain after intranasal drug administration to mice, compared with IV route. In spite of its higher systemic exposure, sertraline distributed to peripheral organs (i.e., lungs) in a considerably lower extent than IV and oral routes.

[0031] This innovation encompasses the significant changes that occur in sertraline biodistribution when it is administered by intranasal route, comparatively to IV or oral administrations.

[0032] In a particular embodiment, the present invention allows a sustained drug distribution to the brain and a reduction of drug levels on peripheral organs, namely lungs, that will, hence, reduce the side effects of sertraline.

[0033] In a preferred embodiment, the invention discloses a composition comprising a drug highly binding to plasma proteins for use in the treatment of central nervous system disorders, characterized in that the route of administration is intranasal.

[0034] In a further embodiment, the invention comprises a composition wherein the drug highly binding to plasma proteins is a selective serotonin reuptake inhibitor or a nonsteroidal antiinflammatory drug.

[0035] In a further embodiment, the invention comprises a composition, wherein the drug highly binding to plasma proteins binds at a level of at least 90% of binding to plasma proteins, preferably at least 95%, most preferably at least 98%, even more preferably 99%.

[0036] In a further embodiment, the invention comprises a composition wherein the selective serotonin reuptake inhibitor is administered at a dose suitable for humans.

[0037] In a further embodiment, the invention comprises a composition wherein the selective serotonin reuptake inhibitor is sertraline or piroxicam or a combination thereof.

[0038] In a further embodiment, the invention comprises a composition, wherein intranasal administration is single or multiple administration. [0039] In a further embodiment, the invention comprises a composition, wherein the disease is a psychiatric disease, in particular depression, post-traumatic stress, obsessive-compulsive disorder, panic disorder, premenstrual dysphoric disorder, social anxiety disorder or any other central nervous system disease.

[0040] In a further embodiment, the invention comprises a composition, wherein the disease is characterized be neuroinflammation, including neurodegenerative disease, a neuropsychiatric disease, a neurological disorder or a tumor, particularly a brain tumor.

[0041] In a particular embodiment, the present invention encompasses a pharmaceutical formulation comprising the composition of any one of the previous embodiments.

[0042] In a particular embodiment, the present invention encompasses a pharmaceutical formulation for use as an intranasal medicament.

[0043] In a particular embodiment, the present invention encompasses a pharmaceutical formulation in the form of a thermoreversible hydrogel, a suspension, a powder, a liposome, an emulsion, a microemulsion, a nanoemulsion, a nanoparticle, a nanostructured lipid carrier, a gel, a nanosystem, a chitosan nanoparticle, an alginate-chitosan nanoparticle or other nanoparticle-based formulations and/or a combination thereof.

[0044] In a particular embodiment, the present invention encompasses a pharmaceutical formulation for brain sustained delivery of a drug highly binding to plasma proteins, particularly sertraline.

[0045] In a particular embodiment, the present invention encompasses a pharmaceutical formulation further comprising one or more elements from the list: an excipient, a coating, an additive, a lubricant, a filler, a colouring agent, a flavouring agent or a combination thereof.

[0046] In a final embodiment, the present disclosure comprises a device for intranasal administration of the composition or formulation of the previous embodiments further comprising means for administrating the composition to a patient.

Brief Description of the Drawings

[0047] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0048] Figure 1 depicts the structure of sertraline hydrochloride.

[0049] Figure 2 represents the temporal evolution of sertraline concentrations in plasma (A), brain

(B) and lungs (C) after intranasal (IN) and intravenous (IV) administration to mice, at the dose of 4.87 mg/kg (n = 5, per time point). Results are represented as mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0050] Figure 3 represents the evolution of brain-to-plasma (A) and lung-to-plasma (B) ratios of sertraline at 5, 15, 30, 60, 120 and 180 min after intranasal (IN) and intravenous (IV) administrations. Results are expressed as mean ± standard error of the mean (n = 5). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0051] Figure 4 represents the temporal evolution of sertraline concentrations in plasma (A), brain (B) and lungs (C) after intranasal (IN, 4.87 mg/kg) and oral administration (10 mg/kg) to mice (n = 5, per time point). Results are represented as mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0052] Figure 5 represents the evolution of brain-to-plasma (A) and lung-to-plasma (B) ratios of sertraline at 5, 15, 30, 60, 120 and 180 min after intranasal (IN) and oral (IV) administrations. Results are expressed as mean ± standard error of the mean (n = 5). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0053] Figure 6 represents the temporal evolution of piroxicam concentrations in plasma (A), brain (B) and lungs (C) after intranasal (IN) and intravenous (IV) administration to mice, at the dose of 5 mg/kg (n = 3-4 per time-point). Results are represented as mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0054] Figure 7 represents the evolution of brain-to-plasma (A) and lung-to-plasma (B) ratios of piroxicam at 5 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after intranasal (IN) and intravenous (IV) administrations. Results are expressed as mean ± standard error of the mean (n = 3-4). ****p<0.0001.

[0055] Figure 8 represents the cell viability (%) of RPMI 2650 cells after incubation with sertraline for 24h (from 0.01 to 12.5 pM). Data are represented as mean ± standard deviation (n=3). Ctrl: control group, without treatment *p<0.05 and ***p<0.001 in relation to the control, analysed by one-way analysis of variance (ANOVA), with multiple comparison test.

Detailed Description

[0056] The inventors have unexpectedly identified a novel drug biodistribution behaviour for a CNS-acting drug administered by means of intranasal route. More specifically, the intranasal administration of sertraline attained higher systemic exposure and a slower distribution into the brain and peripheral tissues (lungs) relatively to IV administration. [0057] Furthermore, brain/plasma ratios were comparable between intranasal and oral routes, thereby anticipating similar therapeutic effects. The lung/plasma ratio of intranasal sertraline was lower than oral formulation, which may lead to decreased side effects. The decreased lung/plasma ratio observed after intranasal administration relatively to IV or oral routes, contributes to the improvement of drug safety.

[0058] The slower distribution of sertraline into the brain herein described, does not compromise its therapeutic effect since, in steady state, constant levels are achieved.

[0059] In one embodiment, it was herein discovered that the absolute bioavailability of sertraline is higher than 100 %, as a result of its slower incorporation.

[0060] The solution provided by the present invention overcomes the problem of plasma protein saturation that occurs with IV bolus, which, in turn, would accelerate and enhance drug distribution into brain and lungs. Since sertraline is highly bound to plasma proteins (around 98%), the authors postulate that intranasal administration allows a slower drug access to the bloodstream, avoiding saturation of plasma protein binding sites and, hence, slowing down drug distribution. In opposition, when intravenously administered, sertraline saturates plasma protein binding sites quickly, and its free fraction increases, as well as its distribution to highly irrigated organs (e.g. brain and lungs).

[0061] In one embodiment, the invention relates to a sustained delivery of sertraline to the brain after intranasal instillation, yielding drug concentrations more stable and avoiding peaks-to-troughs fluctuations that occur when a drug quickly attains Cmax (e.g., following IV injection). This behaviour is very important in chronic treatments of CNS diseases, such as depression and others.

[0062] In one embodiment, the invention promotes a considerable decrease of sertraline exposure in lungs, comparatively to IV and oral routes. Therefore, less side effects are expected to occur with the intranasal instillation herein proposed.

[0063] In an embodiment, multiple administrations of the intranasal formulation in chronic CNS diseases, such as depression, neurological and neurodegenerative diseases, will reduce peak-to- trough fluctuations of steady-state brain concentrations, thereby decreasing the potential to develop central toxic and subtherapeutic effects.

[0064] The reduced distribution of intranasally administered sertraline to peripheral tissues also envisions fewer peripheral side effects.

[0065] In one embodiment, smaller doses of sertraline, preferably comprising a reduction of about 50% of the dose comparatively to the usual dosage for oral administration, are administered by intranasal route, therefore reducing adverse side effects, hepatic first-pass effect and the potential to develop DDIs. [0066] In an embodiment, a nasal thermoreversible hydrogel formulation composed of 18% Pluronic F-127 and 0.2% Carbopol® 974P was used. The same hydrogel had been previously applied by team members to deliver antiepileptic drugs from the nasal cavity into the brain [13, 14, 44, 45]. This background work evidenced that the intranasal administration of CNS-acting drugs gives rise to plasma and brain pharmacokinetic profiles that are similar to those achieved after IV injection [36, 37]. Nonetheless, in some cases, intranasal administration even increased Cmax and decreased tmax values in brain, comparatively to IV and oral routes, allowing a faster action of the drug, attributed to direct nose-to-brain transport [13, 14,38].

[0067] The present invention encompasses the following disorders: obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, premenstrual dysphoric disorder, social anxiety disorder, Alzheimer disease, Parkinson disease, neuroinflammatory diseases, Neuromyelitis optica (NMO), Anti-myelin oligodendrocyte glycoprotein antibody disorder (MOG), Autoimmune encephalitis, Transverse Myelitis, Optic neuritis, Neurosarcoidosis, among other related disorders.

[0068] The following examples are included to demonstrate certain embodiments of the invention.

[0069] The data herein provided were obtained from a randomized pharmacokinetic pre-clinical study in healthy male CD-I mice. Mice were treated with intranasal sertraline loaded into a thermoreversible gel, an aqueous IV sertraline solution or a Zoloft® suspension, at the doses of 4.87 mg/kg for intranasal and IV routes and 10 mg/kg for oral route.

[0070] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0071] It should be appreciated by those of skill in the art that the findings disclosed in the examples represent techniques discovered by the inventors. However, in light of the present disclosure, it should be noted that many changes can be made in the specific embodiments which are disclosed and still obtain an equivalent or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1 - Intranasal vs. IV sertraline administration

[0072] This pre-clinical in vivo experiment aimed to explore the potential of the intranasal route to administer sertraline, evaluate its systemic bioavailability and compare its brain and lung pharmacokinetic profiles with those observed after IV injection at the same single dose (4.87 mg/kg). Male CD-I mice (20-25g) were randomly divided into two groups: one received sertraline through intranasal administration and the other one was intravenously administered. [0073] The thermoreversible hydrogel composed of 18% Pluronic F-127 and 0.2% Carbopol® 974P was loaded with sertraline and 25 pL were administered into the right nostril using a polyurethane tube (24 G, 19 mm) coupled to a 1 mL syringe.

[0074] For IV administration, the same dose was injected into the lateral tail vein using an insulin syringe (27 G, 1.0 mL).

[0075] At 5, 15, 30, 60, 120 and 180 minutes post-administration (n = 5 per time point) by intranasal or IV routes, blood was collected into heparinized tubes while brain and lungs were excised, gently washed with sodium chloride 0.9% solution, dried with sterile compresses and weighed. Blood was centrifuged to collect plasma while tissues were homogenized with 4 mL of sodium phosphate buffer (0.1 M, pH 5.0) per g of tissue, as previously reported [13, 14, 43]. All samples were stored at -80 °C until appropriate treatment and subsequent analysis with resort to a validated high performance liquid chromatography (HPLC) with diode-array detection (DAD). The method was validated in plasma (0.08 - 3 pg/mL), brain (0.04 - 1.5 pg/mL) and lung (0.5 - 18 pg/mL) according to the international bioanalytical method validation guidelines. The technique demonstrated intra- and inter-day accuracy (-10.65 to 10.55%) as well as intra- and inter-day precision (2.34 to 13.14%) for all matrices.

[0076] The mean experimental concentration versus time profiles were plotted in plasma, brain and lungs and, then, submitted to non-compartmental pharmacokinetic analysis, using the WinNonlin software (version 5.2 Pharsight Co).

[0077] The estimated pharmacokinetic parameters included the maximum concentration (Cmax) of sertraline in plasma and tissues, tmax, the area under the concentration-time curve from time zero to the time of last measurable concentration (AUCt), and from time zero to infinity (AUCinf) and the apparent elimination rate constant (Kel) and the mean residence time (MRT).

[0078] The bioavailability of the intranasally administered drug was calculated by comparing the AUCt between intranasal and IV routes, expressed as a percentage of IV bioavailability. The brain bioavailability of sertraline, corresponding to the ratio between the AUCt in brain after intranasal and IV administrations, was also estimated. Plasma-to-tissue ratios were also determined to compare the affinity of sertraline for each organ in relation to its systemic exposure.

[0079] In order to assess the tendency of the drug to reach the brain after intranasal administration, the drug targeting efficiency (DTE) and direct transport percentage (DTP) were calculated according to reference [43].

[0080] Mean sertraline concentrations and standard error of the mean (n = 5) obtained at each time point in plasma, brain and lung after single dose administration by both routes are depicted in Figure 2. The corresponding pharmacokinetic parameters are presented in Table 1, while tissue-plasma ratios are chronologically depicted in Figure 3.

Table 1. Pharmacokinetic parameters of sertraline in plasma, brain and lung tissues following its intravenous (IV) and intranasal (IN) administration at the dose of 4.87 mg/kg to mice.

Pharmacokinetic ^{piasma Brain Lun} 8 ^a Parameters were estimated using the mean concentration-time profiles obtained from five different animals per time point (n = 5). ^b Values expressed in pg/g; ^c Values expressed in pg.min/g; ^d Absolute intranasal bioavailability (F) was calculated based on AUCt values; AUCextrap, extrapolated area under drug concentration-time curve; AUCinf, area under drug concentration-time curve from time zero to infinity; AUCt, Area under the concentration-time curve from time zero to the last quantifiable drug concentration; Cmax, Maximum peak concentration; kel, Apparent elimination rate constant; MRT, Mean residence time; tl/2el, Apparent terminal elimination halflife; tmax, Time to achieve the maximum peak concentration.

[0081] The first surprising finding regards the plasma concentrations observed at 5 and 15 min after IN administration, which are considerably higher than those of IV injection (p<0.01 and p<0.001, respectively, Figure 2A). Afterwards, plasma pharmacokinetic profiles overlap for both routes of administration. Consequently, the absolute bioavailability of sertraline after in situ nasal gel administration is higher than 100% (166.33 %, Table 1). This was completely unexpected and the inventors justify this novelty due to the high binding of sertraline to plasma proteins. Indeed, when administered by IV route, drug molecules are instantly available in plasma, saturating binding locals of plasma proteins. This will increase the free drug fraction, which will, then, quickly cross biological membranes, including the BBB and distribute to the most irrigated tissues, namely the brain and the lungs. Observing Figures 2B and 2C, it is evident that the concentrations in brain and lungs are considerably higher after IV injection, supporting the present disclosure. [0082] The second surprising result regards the sustained brain delivery of sertraline after intranasal administration (Figure 2B). Despite the fast decrease of plasma concentrations, sertraline crosses the BBB slowly, attaining Cmax in the brain at 60 min post-administration (vs 15 min after IV injection). In chronic depression treatment, it is more important to maintain stable levels in the brain than peaks-to-troughs, which increase drug level fluctuations and consequently enhance response variability and hamper drug titration.

[0083] There was no direct nose-to-brain delivery of sertraline, as evidenced by the DTE of 24.45 % and DTP of 13.43 % (Table 1).

[0084] The temporal evolution of brain-to-plasma ratios depicted in Figure 3A demonstrates an increase of ratios with post-administration time. Until to 60 min, brain-to-plasma ratios of the intranasal route are statistically inferior to those of IV administration. From then on, the ratios observed after intranasal instillation become superior to those of IV injection. In fact, at 180 min post-administration, the brain-to-plasma ratio of the intranasal route is more than 3-fold of the IV route (p<0.001). This corroborates that sertraline reaches the brain slowly, but its delivery is maintained for a longer period of time. The lower Kel observed with the intranasal route (0.0047 vs 0.0142 min-1), and consequently, its longer tl/2 (148.32 vs 48.80 min, Table 1), highlight the sustained brain delivery of sertraline from the bloodstream.

[0085] Another surprising effect of the present invention regards the disposition of sertraline in the lungs after intranasal administration. Indeed, as aforementioned, it is known that sertraline tends to accumulate in the lungs and promote severe respiratory adverse effects.

[0086] In the present disclosure, it was found that the intranasal route not only sustains sertraline delivery into the brain, but also reduces lung exposure considerably, relatively to IV injection (AUCt of 217.655 vs 973.805 pg.min/mL, Table 1). Consequently, fewer side effects are expected to occur.

[0087] The present invention provides the technical effect that sertraline biodistribution is substantially modified when intranasally administered, allowing an absolute bioavailability higher than 100% and a sustained delivery into the brain, in addition to a slower and less extensive distribution to the lungs.

Example 2 - Intranasal vs. oral sertraline administration

[0088] Since sertraline is currently marketed as oral formulations, its pharmacokinetic profiles in plasma, brain and lung after intranasal administration to male CD-I mice were also compared with those obtained after oral administration of a Zoloft® suspension (20 mg/mL).

[0089] The experimental protocol was the same as previously described in Example 1. Oral administration, performed instead of IV injection, required the dilution of Zoloft suspension with saline (0.8 mg/mL) which was, then, administered by oral gavage (12.5 mL/kg) at the dose of 10 mg/kg.

[0090] Samples were collected, prepared and analysed as in Example 1 and the same pharmacokinetic parameters were determined. Figure 4 represents the mean dose-normalized sertraline concentrations and standard error of the mean (n = 5) obtained at each time point in plasma, brain and lungs after single oral and intranasal administrations, respectively. Figure 5 represents the variation of tissue-plasma ratios alongside with post-administration time. The parameters of the dose-normalized exposure of sertraline, estimated for oral and intranasal administrations, are summarized in Table 2.

Table 2. Dose-normalized pharmacokinetic parameters of sertraline in plasma, brain and lung after its intranasal (IN, 4.87 mg/kg) and oral (10 mg/kg) administration to mice. ^a Parameters were estimated using the mean concentration-time profiles obtained from five different animals per time point (n = 5). AUCinf, Area under the concentration time-curve from time zero to infinite normalized per administered dose; AUCt, Area under the concentration timecurve from time zero to the last quantifiable drug concentration; Cmax, Maximum peak concentration.

[0091] Dose-normalized plasma concentrations are comparable between intranasal and oral routes, except for 15 and 60 min, where statistical differences were identified (Figure 4A, p<0.05). Consequently, dose-normalized plasma AUCt were almost the same for both administration routes (Table 2).

[0092] The present disclosure is particularly relevant for future human applications, namely New Drug Application (NDA) biodistribution and bioequivalence studies. Indeed, since the systemic exposure of intranasal sertraline is similar to that of the marketed formulation (oral Zoloft®), bioequivalence is highly likely to occur.

[0093] Regarding brain disposition, the pharmacokinetic profiles are parallel, even though the concentrations of intranasal instillation are slightly lower (Figure 4). Nonetheless, dose-normalized Cmax values are similar and no statistical differences between brain-to-plasma ratios were found (with exception of the post-administration time of 180 min, Figure 4B). [0094] It was herein also discovered that the lung exposure of sertraline is decreased after intranasal instillation, comparatively with oral gavage [44.69 vs 66.43 (pg.min/g)/(mg/kg), Table 2]. This corroborates the finding of Example 1, supporting that lung accumulation of sertraline is reduced with the intranasal in situ gel administration, leading to decreased side effects.

[0095] This invention claims that the lung exposure of sertraline decreases considerably after intranasal instillation, comparatively to marketed oral routes. Therefore, fewer side effects are expected to occur.

Example 3 - Intranasal vs. IV piroxicam administration

[0096] This pre-clinical in vivo experiment aimed to explore the potential of the intranasal administration of an anti-inflammatory drug, piroxicam, and evaluate its systemic bioavailability, comparing brain and lung pharmacokinetic profiles with those obtained after IV injection in the same single dose (5 mg/kg). Male CD-I mice (20-25g) were randomly divided into two groups: one received piroxicam by intranasal administration and the other was intravenously administered.

[0097] The thermoreversible hydrogel composed of 18% Pluronic F-127 and 0.2% Carbopol® 974P was loaded with piroxicam and 0.5 pL/g were administered into the right nostril using a polyurethane tube (24 G, 19 mm) coupled to a 1 mL syringe.

[0098] For IV administration, the same dose was injected into the lateral tail vein using an insulin syringe (27 G, 1.0 mL).

[0099] At 5 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h post-administration by intranasal or IV routes, blood was collected into heparinized tubes while brain and lungs were excised, gently washed with sodium chloride 0.9% solution, dried with sterile compresses and weighed. Blood was centrifuged to collect plasma, while brain and lung tissues were homogenized with 3 or 4 mL of sodium phosphate buffer (0.1 M, pH 5.0) per g of tissue, respectively. All samples were stored at -80 °C until appropriate treatment and subsequent analysis with resort to a HPLC-DAD technique. The method was validated in plasma (0.08 - 24 pg/mL), brain (0.081 - 30 pg/g) and lung (2.0 - 400 pg/g) according to the international bioanalytical method validation guidelines. The technique demonstrated to be accurate (-13.34 - 11.38%) and precise (1.43-12.55%) in all matrices.

[00100] The mean experimental concentration versus time profiles in plasma, brain and lungs were plotted and, then, submitted to non-compartmental pharmacokinetic analysis, using the WinNonlin software (version 5.2 Pharsight Co).

[00101] Cmax, tmax, AUCt, AUCinf, Kel and MRT were calculated for piroxicam.

[00102] The bioavailability of intranasally administered piroxicam was calculated as reported in Example 1 as well as the brain bioavailability, which corresponds to the AUCt ratio in brain after intranasal and IV administrations. Plasma-to-tissue ratios were also determined to compare the affinity of piroxicam to each organ in relation to its systemic exposure.

[00103] DTE and DTP were calculated as indicated in Example 1.

[00104] Mean piroxicam concentrations and standard error of the mean obtained at each time point in plasma, brain and lung after single dose administration by both routes are depicted in Figure 6. The corresponding pharmacokinetic parameters are presented in Table 3, while tissueplasma ratios are chronologically depicted in Figure 7.

Table 3. Pharmacokinetic parameters of piroxicam in plasma, brain and lung tissues following their intranasal (IN) or intravenous (IV) administration to mice (5 mg/kg). ^a Parameters were estimated using the mean concentration-time profiles obtained from different animals per time point (n = 3-4). ^b Values expressed in pg/g; ^c Values expressed in pg.min/g; ^d Absolute intranasal bioavailability (F) was calculated based on AUCt values; AUCextrap, Extrapolated area under the drug concentration time-curve; AUCinf, Area under the concentration time-curve from time zero to infinite; AUC _t , Area under the concentration time-curve from time zero to the last quantifiable drug concentration; C _max , Maximum peak concentration; k _e i, Apparent elimination rate constant; ti/2ei, Apparent terminal elimination half-life; t _max , Time to achieve the maximum peak concentration.

[00105] Plasma concentrations observed after intranasal piroxicam administration were always higher than those observed for IV injection, with statistically significant differences observed at 5 min post-dosing (p<0.01, Figure 6A). Consequently, the absolute bioavailability of piroxicam after in situ nasal gel administration was higher than 100% (132.04 %, Table 3), corroborating the findings registered for sertraline (Example 1). Piroxicam binds 98-99% to plasma proteins [46] and may saturate protein binding sites when administered in bolus injection. Moreover, similarly to sertraline, piroxicam also exhibited a sustained brain delivery after intranasal administration (Figure 6B), with a longer half-life time after intranasal instillation (8.2617 h vs 2.4071 h, Table 3). The temporal evolution of brain-to-plasma ratios are depicted in Figure 7 A. With exception of the 6 h sampling time, intranasal administration afforded equal or higher values in comparison to IV injection, highlighting the sustained delivery into the brain as herein postulated. Residual direct nose- to-brain delivery was detected, as evidenced by the DTE of 108.49 % and DTP of 7.83 % (Table 3).

[00106] Piroxicam exposure in the lung was slightly higher after intranasal instillation (Figure 6C), but statistically significant differences (p<0.05) were registered only at 5 min post-dosing (6.3610 pg.h/g vs 2.7379 pg.h/g). Moreover, lung-to-plasma ratios were higher 5 min after intranasal administration than IV injection, but were statistically lower 24 h post-administration. Furthermore, a longer half-life time was observed in lung, after IV dosing (6.02 vs. 4.90 h, Table 3). These findings suggest that piroxicam distribution to peripheral tissues (e.g., lung) is longer after IV injection, increasing the risk of drug accumulation and peripheral side effects.

[00107] The present example corroborated the technical effect that biodistribution is substantially modified when a drug binding to plasma proteins in an extent of 99% is intranasally administered, allowing an absolute bioavailability higherthan 100% and a sustained delivery into the brain with reduced accumulation in peripheral tissues.

Example 4 - In vitro cell culture to assess the impact of sertraline in the viability of nasal cells

[00108] The human tumour cell line from nasal septum squamous epithelium (RPMI 2650, ECACC 88031602) was used for viability experiments. The cells were cultured in Eagle's Minimum Essential Medium (EMEM, M2279) supplemented with 2 mM glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin mixture and 10% heat-inactivated fetal bovine serum (Gibco Life Technologies, ThermoFisher Scientific, Waltham, MA, USA). Cells were grown in T75 flasks (Orange Scientific, Braine-I' Alleud, Belgium), passaged twice a week using a 0.25% trypsin-EDTA solution and cultured at 37 °C in 5% CO2 and 95% relative humidity. All assays were performed with RPMI 2650 cells with passage numbers below 30.

[00109] The influence of sertraline on cell viability was determined by the Alamar blue assay. RPMI 2650 cells were seeded into 96-well plates (Orange Scientific Braine-l'Alleud, Belgium) at a density of 6.0xl0 ⁴ cells/well and cultured for 24 hours in a humidified incubator at 372C in 5% CO2. After removing the culture medium, 200 pL of fresh medium without (control cells) or sertraline in different concentrations (0.01-25 pM) were added to cells and incubated for 24 hours. Afterwards, treatment solutions were removed and fresh medium with 10% Alamar Blue solution (125 mg/mL) was added, followed by incubation for 3 hours. A Biotek Synergy HT microplate reader (Biotek Instruments®, Winooski, VT, USA) was used for fluorescence measurements at 530/590nm (excitation and emission wavelengths).

[00110] Equation 1 was applied for the calculation of cell viability: where Fldrug, Flblank and Flcontrol correspond to the mean fluorescence in wells after incubation with sertraline, empty wells and wells without any treatment, respectively. The experiment was performed three times (n = 3) with three replicates for each condition. The cut-off line considered that drug concentrations compromise cell viability when values below 85% are observed.

[00111] The experimental results (Figure 6) revealed that cell viability was maintained above 85% after 24 h of incubation of RPMI 2650 cells with sertraline at 12.5pM, although the highest concentration had statistical differences comparatively to the control.

Example 5 - Validation of bioanalytical method to quantify sertraline in plasma, and brain and lung homogenates

[00112] To quantify sertraline in plasma, brain and lung samples collected from pharmacokinetic studies (Examples 1 and 2) with accuracy and precision, a HPLC-DAD technique was developed and fully validated, in accordance to international guidelines of the European Medicines Agency and FDA. Firstly, samples were treated and sertraline was extracted by means of a double liquid-liquid extraction with dichloromethane.

[00113] Briefly, aliquots of blank plasma (100 pL), brain homogenate (200 pL) and lung homogenate (100 pL) were thawed at room temperature and centrifuged at 12.045 g for 2 min. Then, the samples were spiked with 10 pL of sertraline working solutions and 10 pL of internal standard (IS) working solution. Thereafter, 1 mL of dichloromethane was added, followed by 1 min vortex stirring and 5 min of centrifugation at 12.045 g. The bottom organic layer was collected into a glass tube and 1 mL of dichloromethane were re-added to the remaining aqueous layer. The combined organic phases were evaporated to dryness under a nitrogen stream at 60°C. Lastly, the residue was reconstituted with 100 pL of phosphate buffer (20 mM pH 3.8) and acetonitrile mixture (60:40, v/v). The glass tube was sealed and vortexed during 1 min, and the mixture was collected into a Costar® Spin-X® centrifugal filter (Corning, Inc., NY, USA), which was centrifuged during 2 min at 12.045 g. The filtrate was transferred into a vial, injected into the HPLC system and subjected to analysis.

[00114] The chromatographic separation of sertraline and the IS was accomplished in 14 min using a reversed-phase LiChroCART® Purospher® Star C18 column (55 mm x 4 mm, 3 pm particle size; Merck KGaA) and isocratic elution (1.0 mL/min) with a mobile phase composed of phosphate buffer 20 mM pH 3.8 and acetonitrile (65:35, v/v). Oven temperature was set at 40°C and the injection volume was 20 pL. Detection wavelengths were set at 227 nm and 245 nm for sertraline for the IS, respectively. [00115] For mouse plasma, intra-day values of precision [given by coefficient of variation (CV)] varied between 6.47 to 11.1%, while accuracy (given by Bias %) varied between -3.67 to 1.90%. The inter-day values of CV varied between 4.71 to 13.1% and Bias varied between -2.37 to 5.38%. In brain homogenate, the intra-day values of CV varied between 3.53 to 12.6% and Bias varied between -7.37 to 10.5%. The inter-day values of CV varied between 4.29 to 9.20% and Bias varied between - 1.17 to 9.76%. Lastly, for lung homogenate, the intra-day values of CV varied between 2.32 to 8.32% and Bias varied between -10.6 to 8.26%. The inter-day values of CV varied between 5.14 to 10.6% and Bias varied between -6.23 to 2.61%.

[00116] Intra-day and inter-day values of Bias for the lower limit of quantification (LLOQ) in plasma were 0.46% and 5.38%, respectively. Accordingly, intra-day CV was 6.47%, and inter-day CV was 7.07%. Regarding homogenized brain, the intra-day and inter-day values of Bias for the LLOQ were -2.46% and 9.76%, respectively. While the intra-day and inter-day CV were 10.9% and 4.29%. Regarding homogenized lung, intra-day and inter-day values of Bias for the LLOQ were 8.26% and 2.61%, respectively, while intra-day and inter-day CV were 2.34% and 5.14%.

[00117] Overall, the acceptance criteria for accuracy and precision were fulfilled because the Bias for quality control samples was within 15% of the nominal values, except for the LLOQ, which was within 20% of the nominal value. Precision acceptance criteria were also met because the CV for the quality control samples did not exceed 15%, with the exception of the LLOQ, which did not exceed 20%.

[00118] Any drug which have the brain as target organ and that are binding to plasma protein at least 95% can be herein included.

[00119] The examples are herein included for the purpose of illustration-only and are not intended to be limiting of the invention or any embodiment thereof, unless so specified.

[00120] In an embodiment, the present invention is intended to be applied to humans.

[00121] In another embodiment, the present invention is applied to other CNS-acting drugs besides sertraline, specifically CNS drugs that are highly bound to plasma proteins (>90%).

[00122] In another embodiment, the present invention is applied to other drug formulations, including suspensions, powders, nanosystems (liposomes, micro/nanoemulsions, chitosan nanoparticles, alginate-chitosan nanoparticles, poly (lactic-co-glycolic acid) -chitosan, thiolate chitosan nanoparticles, nanostructured lipid carriers, among others).

[00123] In another embodiment, the present invention fits other therapeutic unmet needs as future in vivo medicine application, such as neurodegenerative diseases, neuropsychiatric and neurological disorders, brain tumours. [00124] The above-described embodiments are combinable.

[00125] The following claims further set out particular embodiments of the disclosure.

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