Patent Application
20200222323
The present invention relates to double layered lipid vesicles, preferably liposomes, containing adrenaline incorporated inside them, for use in the treatment of cardiac emergencies.
The invention also relates to a pharmaceutical composition comprising said lipid vesicles, preferably in the form of an injectable liquid solution, in particular for parenteral administration during cardiac emergencies.
Adrenaline (or epinephrine) is a hormone and a neurotransmitter belonging to the class of compounds called catecholamines, since their structure comprises both an o/i/70-dihydroxy benzene (whose chemical name is catechol) and an amine group.
Physiologically, it is secreted by chromaffin cells of the medullar region of the adrenal gland following stimulation by the sympathetic nervous system as a consequence of strong emotions, fear in particular, and in general in situations in which a “fight or flight” reaction may be necessary, i.e. where there may be the need for an escape, a fight, or in any event an increase in physical activity. Precisely for this reason, the effects of adrenaline at a systemic level include: gastrointestinal relaxation, a dilatation of the bronchi, an increase in heart rate and stroke volume (and consequently cardiac output), a diversion of blood flow towards muscles, the liver, the heart and the brain and an increase in glycaemia. The cell receptors that bind adrenaline and transmit the signal mediated by it are called adrenergic receptors.
Since the effects of adrenaline are varied and very intense, and involve numerous regions of the human body, the molecule has been used for some time as a drug in cardio-pulmonary resuscitation, typically by
parenteral administration: its action is in fact fundamental in the event of a cardiac arrest, since it is capable of stimulating myocardial contraction and resumption of the heartbeat.
The reason for using adrenaline lies in its action on a-adrenergic receptors, which trigger arteriolar vasoconstriction in the periphery (fundamental for restarting spontaneous circulation) and vasoconstriction of the veins, with a consequent redistribution of blood to vital organs. Adrenaline thus diverts blood flow away from the periphery to central circulation, protecting vital organs from ischaemia through the redistribution of the volume. This action leads to an increase in the coronary perfusion pressure, which is a prognostic factor for the restoration of spontaneous circulation. Moreover, since adrenaline does not penetrate through the blood-brain barrier, the cerebral perfusion pressure increases indirectly, as a result of the volume redistribution.
However, as it is a non-selective adrenergic receptor agonist, adrenaline exerts its action both on a receptors and β receptors, which have a differential distribution in the body and mediate different effects. The action of adrenaline on β1 receptors provokes vasodilation and activation of the β1 receptors of the heart, which mediate positive inotropic and chronotropic effects. Thus, adrenaline increases myocardial contraction, thereby increasing the oxygen demand of a heart already deficient in oxygen due to the cardiac arrest.
Furthermore, since a-adrenergic receptors are also expressed at the level of brain microvessels, a vasoconstriction occurs at the level of brain microcirculation, which exacerbates the ischaemia after the return of spontaneous circulation and increases the risk of neurological damage. Moreover, although the use of adrenaline favours the return of spontaneous circulation, it has been observed that in the event of a prolonged cardiac arrest, patients' likelihood of survival decreases with the repeated administration of adrenaline.
At present, the guidelines for cardio-pulmonary resuscitation provide for intravenous administration of a solution comprising adrenaline at a dose of 0.014 mg/kg of body weight (corresponding to 1 mg for an adult weighing 70 kg) every 3-5 minutes during cardio-pulmonary resuscitation. The frequency of administration is due to the brief half-life (and hence brief duration of action) of adrenaline in vivo (no greater than 5 minutes), a fact that makes repeated administration necessary in order to maintain the therapeutic concentration in the target tissue.
Repeated administrations exacerbate, in turn, the side effects of adrenaline, leading to a significant reduction both in the duration and quality of the life of patients, above all due to the neurologic deficits induced by the treatment.
The adrenaline solutions commonly available on the market and used in cardio-pulmonary resuscitation contain the active ingredient in concentrations of from 0.15 to 1 mg/ml, sodium chloride as an isotonifier, hydrochloric acid to bring the pH to 2.5 and permit the dissolution of the adrenaline, sodium metabisulphite to prevent oxidation and water for injectable preparations.
In the light of the above, therefore, it appears that the administration of adrenaline during cardio-pulmonary resuscitation can give rise to major side effects, the reason why the relationship between the risks and benefits of treatment with adrenaline is still a subject of debate.
In particular, high doses of adrenaline, useful for an immediate resuscitation effect, and high doses deriving from cumulative administrations increase the severity of neurologic side effects. For this reason, it appears to be of great importance to develop a system for the administration of adrenaline that may enable a controlled release in the body, reducing the number of administrations, the cumulative dose and, accordingly, the side effects.
Liposomes are vesicular structures that are substantially spherical in shape and made up of one or more double layers (bilayers) of phospholipids, whose composition is similar to that of cell membranes. Being biocompatible and nontoxic, liposomes have long been used in medicine as a drug delivery system, i.e. as vehicles for delivering molecules with a pharmacological or biological action into the body. In their simplest structure (unilamellar liposomes), liposomes are formed by a single bilayer of phospholipids; the phospholipids of the outer layer expose the polar heads towards the surrounding environment, whilst the nonpolar tails are turned towards the inside, where they interface with the nonpolar tails of the second lipid layer, whose organisation mirrors the previous one, i.e. the polar heads are turned towards the cavity of the liposome, which delimits an aqueous core. Unilamellar liposomes typically have a diameter comprised between 50 and 500 nm. Multilamellar liposomes, by contrast, are characterised by the presence of various concentric lipid bilayers, separated from one another by aqueous environments (“onion-like” structure). Multilamellar liposomes reach diameters comprised between 500 and 10000 nm.
Given that, as said, an aqueous environment (or more than one aqueous environment) comes to be created inside liposomes, liposomes can incorporate and deliver both hydrophilic molecules (in the aqueous core) and lipophilic molecules (inside the bilayer, between the nonpolar tails of the phospholipids) to the site of action. The preparation techniques and surface modifications enable the production of various liposomes for different applications. In fact, there are different methods for preparing liposomes with different characteristics in terms of size, lamellarity and encapsulation effectiveness. In particular, the encapsulation of drugs can be obtained by passive loading during liposome preparation, without any further steps, or with active loading processes, which usually result in greater efficiency compared to passive methods. Active loading (or remote loading) results in the drug's diffusion inside the empty liposome following the creation of a gradient between the inside and outside. For example, drugs can be encapsulated in response to the transmembrane pH gradient generated by acidic buffers present in the inner core or by dissociable proton-generating salts such as ammonium sulphate. Such procedures are known in the state of the art. For example, in order to generate a transmembrane pH gradient, liposomes can be hydrated with a known pH buffer, then dialysed in excess of another buffer with a different pH, to replace the buffer outside the liposomes. The external buffer is selected so that it will not impart any electrical charge to the molecule; the buffer present in the inner core of the liposome, by contrast, is selected so that it will electrically charge the molecule, once it has passed through the bilayer and entered the liposome. Since the charged molecules cannot diffuse through the lipid bilayer, the molecule remains trapped inside the liposome.
For example, WO20131 14377 describes liposomes comprising an amphipathic base and internally loaded with an antitumour drug (doxorubicin, vincristine, topotecan), as well as a method for preparing them by means of an ammonium gradient.
EP361894 describes a method for preparing liposomes comprising an amphipathic drug in a pH gradient, which is formed by creating an ammonium gradient in this case as well.
WO20121 18376 relates to a method for encapsulating in liposomes poorly water soluble substances, whose solubility was previously increased by association with a complexing agent or a co-solvent. The liposomes thus obtained are effective for delivering drugs to the central nervous system by enabling them to cross the blood-brain barrier.
Similarly, US201402201 12 also describes a system for delivering molecules with low water solubility into the body by means of a formulation comprising a complex between such molecules and cyclodextrins and lipid vesicles internally loaded with such molecules, in a non-complexed form. The molecules that can be delivered with this system are, for example, antitumour or anti-inflammatory drugs.
The task of the present invention is to provide a form for the administration of adrenaline that allows the known drawbacks of the prior art described above to be overcome by means of a formulation that is safer and less harmful for the patients who need it.
The present invention also has the object of providing an effective method for loading adrenaline into double layered lipid vesicles (preferably liposomes) by means of a pH gradient process optimised and developed by the Applicant.
The Applicant has in fact observed that in the prior art no concrete examples or experimental data are reported which demonstrate the effective encapsulation of adrenaline in double layered lipid vesicles. The preparation of adrenaline-containing vesicles according to the known pH gradient techniques known in the art in fact have various drawbacks tied precisely to the characteristics of adrenaline.
The process of the present invention makes it possible to overcome such critical aspects, related to the poor stability of adrenaline, which furthermore has various problems of solubility as well as problems of oxidation/degradation that makes its effective encapsulation in double layered lipid vesicles difficult.
A further object of the invention is to develop a system for the gradual and constant release of adrenaline into the body, which makes it possible to avoid repeated administrations of the molecule within close intervals of time in order to keep the available therapeutic dose substantially constant. Within the scope of this object, therefore, it is intended to provide a system for administering adrenaline, in particular during emergency treatments such as cardio-pulmonary resuscitation, which avoids compromising or damaging coronary and/or cerebral blood circulation.
The invention relates to double layered lipid vesicles, preferably liposomes, containing adrenaline incorporated inside them, for use in the treatment of cardiac emergencies.
The invention relates to a pharmaceutical composition comprising said lipid vesicles, preferably in the form of an injectable liquid solution, in particular for parenteral administration during cardiac emergencies.
The invention also relates to a process for preparing adrenaline-containing lipid vesicles.
In the context of the present invention, the term adrenaline (or epinephrine) means the hormone which, at a physiological level, is produced and secreted by chromaffin cells of the medullar region of the adrenal gland following stimulation by the sympathetic nervous system; the adrenaline encapsulated in the double layered lipid vesicles according to the present invention can be either of natural origin (extracted from humans or other vertebrates) or synthetic origin, prepared by chemical synthesis. “Adrenaline” and “epinephrine” are understood as synonyms and are thus used here interchangeably. Furthermore, “adrenaline” also refers to the salts, derivatives and/or precursors thereof, provided that they are physiologically acceptable and pharmacologically suitable for use in the treatment of cardiac emergencies, even where not expressly indicated. Moreover, in the context of the present invention, “adrenaline” indicates the (−) enantiomer, the (+) enantiomer or the racemic mixture indistinctly. The expression “double layered lipid vesicles” means structures of a size comprised between a few tens and a few hundreds of nanometers, formed by at least one lipid bilayer and enclosing one or more hydrophilic environments within them. In contrast, the one or more environments comprised within each bilayer are hydrophobic, being delimited by the lipids themselves.
The expression “adrenaline-containing vesicles (or liposomes)” means that the adrenaline is encapsulated within the internal aqueous cavity (or in the internal aqueous cavities) delimited by the one or more lipid bilayers.
The double layered lipid vesicles that are most common and most widely used in the medical field, above all as carriers for drugs or other molecules of biological or therapeutic importance, are liposomes. In one embodiment of the invention, therefore, the adrenaline-containing double layered lipid vesicles can preferably be liposomes. As is well known in the art, liposomes are formed by at least one bilayer of one or more phospholipids.
The lipid bilayer of the vesicles described here, preferably liposomes, can consist essentially of one or more saturated phospholipids; in another embodiment, the bilayer can consist essentially of one or more unsaturated phospholipids. More typically, however, the bilayer can consist essentially of a mixture of saturated phospholipids and unsaturated phospholipids; for example, the bilayer can be based on lecithin. As is well known in the art, “lecithin” is a general term that indicates a class of phospholipids present in the majority of animal and plant tissues: lecithin is thus a mixture of phospholipids of various types, which can be isolated from various natural sources or purchased commercially.
In a preferred embodiment, the one or more phospholipids forming the bilayer of the lipid vesicles can be selected from the group consisting of: dipalmitoylphosphatidylcholine (DPPC).
distearoylphosphatidylethanolamine (DSPE),
distearoylphosphatidylcholine (DSPC),
dipalmitoylphosphatidylethanolamine (DPPE),
dipalmitoylphosphorylglycerol (DPPG), distearoylphosphorylglycerol (DSPG), dipalmitoylphosphate (DPPA), distearoylphosphate (DSPA), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), palmitoyl-stearoylphosphatidylcholine (PSPC),
dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), palmitoyl oleoylphosphatidylcholine (POPC), stearoyl-oleoylphosphatidylcholine (SOPC), and combinations thereof.
The lipid vesicles, preferably liposomes, can preferably also comprise cholesterol, which, being a nonpolar molecule, is located at the level of the nonpolar “tails” of the phospholipids, inside the phospholipid bilayer. In lipid vesicles, and in liposomes in particular, cholesterol plays a dual function: it reduces the permeability of the lipid bilayer to solutes and improves the packing of the lipids, thus rendering the structure of the vesicles homogeneous and cohesive.
Although lipid vesicles, and liposomes in particular, have a physiological physicochemical structure, which mimics the cell membrane, their half-life in vivo, following parenteral administration, is rather short, since they are captured by mononuclear cells and phagocytised. Another problem tied to the use of lipid vesicles and of liposomes in particular has revealed to be their brief retention of drugs following parenteral administration in vivo. In order to improve the duration of circulation in vivo and the drug retention capacity, it is possible to modulate the size of the lipid vesicles and liposomes in particular, and their physicochemical characteristics.
In one embodiment of the present invention, therefore, the lipid vesicles, preferably liposomes, can have a mean diameter preferably comprised between 50 and 500 nm, more preferably comprised between 50 and 200 nm, even more preferably comprised between 50 and 100 nm. Double layered lipid vesicles of small dimensions (in particular those having a diameter smaller than 100 nm) are in fact capable of escaping the reticuloendothelial system (RES) with greater efficacy than those of larger dimensions, a fact that prolongs the time they remain in the bloodstream after administration.
In another preferred embodiment, the double layered lipid vesicles, preferably liposomes, can be unilamellar, i.e. made up of only one lipid bilayer, preferably a phospholipid bilayer: unilamellar vesicles in fact have smaller dimensions than multilamellar ones, which typically fall in the range specified above.
Furthermore, another preferred embodiment of the invention envisages that the lipids forming the outer layer of the lipid bilayer can be modified by binding one or more hydrophilic polymers to them. Typically, one or more hydrophilic polymers can be bound to the polar heads of the phospholipids present in the outer layer of the vesicles, preferably liposomes. In other words, the vesicles can bear one or more hydrophilic polymers on their outer surface, typically bound to the polar heads of the phospholipids present on their outer layer.
The presence of hydrophilic polymers on the outer surface of the vesicles imparts to them a longer half-life following parenteral administration. In particular, the one or more hydrophilic polymers prevent the capture of the vesicles by the RES (or at least make it difficult), since they form a sort of outer layer that prevents plasma protein from binding to the vesicles themselves (or at least makes it difficult). The presence of the one or more hydrophilic polymers on the outer surface thus increases the stability of the vesicles, ensuring that they remain in the bloodstream longer following parenteral administration, typically by intravenous injection.
The one or more hydrophilic polymers can preferably be selected from the group consisting of: polyethylene glycol (PEG), polyoxazolines, polyvinylalcohols (PVA), polyglycerol, polyvinylpyrrolidone (PVP), poly-N-hydroxypropylmethacrylamide (polyHPMA), polyaminoacids and combinations thereof.
Furthermore, in a preferred embodiment, the percentage of lipids, preferably phospholipids, bearing a hydrophilic polymer bound to them can be comprised between 4% and 15% (molar) relative to the total lipids forming the vesicle.
More preferably, the hydrophilic polymer can be PEG, without limitations in terms of molecular weight and degree of polymerisation; the PEG can preferably have a molecular weight comprised between 2 and 20 KDa. The hydrophilic polymers can be bound to any lipid forming the outer layer of the vesicles which is chemically suited to this bond.
Typically, the hydrophilic polymers can be bound to phospholipids, at the level of their polar head, or to the cholesterol, at the level of the hydroxyl group; the hydrophilic polymers can preferably be bound to distearoylphosphatidylethanolamine (DSPE); more preferably, the hydrophilic polymer bound to DSPE can be PEG.
The lipid vesicles according to the invention, preferably liposomes, can further have a negative surface charge, which is imparted to them by negatively charged lipids and/or derivatives thereof present in the outer lipid layer of the vesicles themselves. Therefore, the outer lipid layer of the vesicles can comprise one or more negatively charged lipids and/or derivatives thereof (anionic lipids). Anionic lipids that can be present in the vesicles of the invention can for example be selected from the group consisting of phosphoglycerides, phosphatidylserine, phosphatidylglycerol. Among negatively charged lipid derivatives, it is possible to mention, for example, phosphatidylethanolamine conjugated to a ligand, for example a hydrophilic polymer or a directing agent. Phosphatidylethanolamine is a zwitterionic molecule that is electrically neutral but carries a positive charge (localised at the level of the amine group) and a negative charge (localised at the level of the phosphate group). When phosphatidylethanolamine is conjugated to a binder, the amine group that engages in the bond is transformed into an amide group, thus losing the positive charge: conjugated phosphatidylethanolamine thus becomes a negatively charged lipid derivative. Advantageously, the molecule conjugated to phosphatidylethanolamine can be selected between a hydrophilic polymer, as previously described, and a directing agent, i.e. a molecule capable of interacting with a biological target towards which it is desired to direct the lipid vesicle. In addition to phosphatidylethanolamine, other zwitterionic lipids and phospholipids can be conjugated to a binder as described above.
The percentage of negatively charged lipids can preferably be comprised between 1% and 40% (molar) relative to the total lipids forming the vesicles, more preferably comprised between 5% and 30% (molar). The percentage range as regards the content of negatively charged lipids is necessarily very wide, since it depends on various elements, such as, for example, correct loading of drug, stability of the structure and toxicity, which are in turn influenced by various factors. In the formulations described in the examples, the content of anionic lipid content is equal to 21% (molar).
The negative surface charge is another characteristic that increases the efficacy of the vesicles for the use described here, as it ensures that they remain longer in the bloodstream following parenteral administration, typically by intravenous injection.
The double layered lipid vesicles, preferably liposomes, containing adrenaline, salts, derivatives or precursors thereof, have application in the medical field, in particular for the treatment of cardiac emergencies. The use of adrenaline is in fact recommended by numerous international guidelines and commonly employed in clinical practice to deal with situations in which the heartbeat is seriously compromised. The treatment of cardiac emergencies generally falls under the heading of “cardiac (or “cardio-pulmonary”) resuscitation”, since it aims to restart a heart muscle with no beat or a beat that is irregular (arrhythmic) to a point where it does enable a blood output such as to ensure normal circulation.
Cardiac arrest (CA) is a clinical situation characterised by ineffectiveness or absence of cardiac activity, which results in a cessation of blood circulation and breathing. The primary treatment for cardiac arrest is, as mentioned, cardio-pulmonary resuscitation (CPR), which should begin as soon as the cardiac arrest is detected or even suspected, in order to maximise the possibility of patient recovery. In the event of cardiac arrest, the heartbeat could be in conditions of ventricular fibrillation or pulseless ventricular tachycardia (collectively called “shockable” rhythms), or show characteristics of pulseless electric activity or asystole (“non-shockable” rhythms). CPR treatment of both groups of conditions includes chest compression and artificial ventilation, whereas in the event of uneven rhythms an electric shock (defibrillation) is necessary. The objective of CPR is to prevent or decrease the brain damage due to hypoxia by restoring a partial flow of oxygenated blood (usually less than 30% of the normal flow) through an external pumping action and to restore spontaneous circulation. The administration of vasoactive drugs at this stage aims to increase systemic vascular resistance in order to obtain a higher perfusion pressure in the myocardium and in the brain and possibly to facilitate the return of spontaneous circulation.
Therefore, the double layered lipid vesicles, and preferably the liposomes, containing adrenaline, or salts, derivatives or precursors thereof can preferably be used for the treatment of conditions selected from the group consisting of: cardiac arrest, ventricular fibrillation, pulseless ventricular tachycardia, pulseless cardiac electric activity, cardiac asystole and combinations thereof. In particular the vesicles, preferably liposomes, can be used for the treatment of cardiac arrest.
The introduction of adrenaline into the aqueous internal cavity of the vesicles is achieved via a pH gradient process developed by the Applicant. This process enables the encapsulation of pharmaceutically active substances (for example, weak anphipathic bases such as adrenaline) in the vesicles by exploiting the pH difference between the aqueous internal cavity of the vesicles (acidic pH typically comprised between 2 and 6) and the external aqueous solution (alkaline pH typically comprised between 8.5 and 10). Since adrenaline is a weak base, in its non-ionised form it is capable of passing through the lipid bilayer of the vesicles. Then, in the acidic environment of the aqueous internal cavity of the vesicles, the adrenaline molecules undergo a protonation, which prevents their further passage through the membrane and therefore their exit from the vesicle itself. The preparation of the adrenaline-containing vesicles according to the pH gradient technique known in the art has drawbacks tied to the scant stability and solubility of adrenaline. These critical aspects have been overcome with the process of encapsulation of the present invention, which ensures an efficient encapsulation of the adrenaline: by way of approximation, more than 75%, preferably about 90%, of the adrenaline present in the starting solution is encapsulated. The encapsulation efficiency is understood as the ratio between the drug trapped inside the liposomes ×100 and the total drug.
The process according to the present invention enables the preparation of adrenaline-containing double layered lipid vesicles, preferably liposomes, by virtue of a suitable adaptation of the pH gradient technique made by the Applicant.
The process comprises the steps of:
a) preparing a buffer solution with an acidic pH comprised between 2 and 6, preferably comprised between 2.4 and 3.0;
b) adding a solution containing lipids to the buffer solution and maintaining under stirring until a vesicular dispersion is formed; c) sonicating the vesicular dispersion for a time comprised between 60 and 200 seconds, preferably between 100 and 150 seconds;
d) adding at least one antioxidant substance suitable for pharmaceutical use;
e) adding a basic solution to the vesicular dispersion until reaching a pH of about 9, preferably comprised between 8 and 9, even more preferably comprised between 8.7 and 9.0;
f) adding adrenaline and maintaining the dispersion under stirring for a time comprised between 5 and 15 hours, preferably between 10 and 13 hours, to enable the diffusion of the adrenaline into the lipid vesicles.
The buffer solution with an acidic pH is preferably obtained from citric acid and disodium hydrogen phosphate or from glycine and hydrochloric acid. In one embodiment the buffer solution is heated to a temperature comprised between 50 and 80° C., preferably between 60 and 70° C., prior to the addition of the solution containing the lipids.
The solution containing lipids is prepared by mixing the lipids in an organic solvent, selected, for example, from chloroform, dichloromethane, ethanol, methanol and diethyl ether.
The lipids are preferably as described above.
The antioxidant substance added after sonication is selected from: sodium metabisulphite, potassium metabisulphite, sodium sulphite, sodium thiosulphate, methionine, monothioglycerol, a-tocopherol, ascorbic acid, citric acid, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT) and mixtures thereof.
The antioxidants are used in a nontoxic concentration, which is such as to effectively prevent oxidation of the adrenaline: this concentration varies according to the selected antioxidant. By way of example, the maximum concentration of BHT for intravenous administration is 0.005 mg/ml and the maximum concentration of monothioglycerol for intravenous administration is 10 mg/ml. In a preferred embodiment, the antioxidant can be sodium metabisulphite: the maximum concentration of sodium metabisulphite for intravenous administration is 10 mg/ml.
The presence of the antioxidant prevents the oxidation of the adrenaline subsequently added into the vesicular dispersion.
The basic solution that is added in order to obtain a basic pH is a solution of a strong base selected from NaOH, KOH or the like.
Steps a)-f) of the process of the present invention are preferably performed in the order described.
For example, it is preferable to add at least one antioxidant substance suitable for pharmaceutical use to the starting vesicular dispersion (comprising the empty lipid vesicles) after the sonication and before step e) of adding the basic solution; In fact, if the at least one antioxidant substance was added after the basic solution, it could induce a substantial modification of the pH, consequently undermining the pH gradient necessary for an efficient loading of the adrenaline into the double layered lipid vesicles.
The adrenaline is preferably added in step f) after alkalinisation of the dispersion (step e)), in order to prevent the degradation of the adrenaline due to oxidation.
In a preferred embodiment of the invention, in step f) the adrenaline is added in an amount such as to obtain a lipids/adrenaline weight ratio of less than 15, preferably comprised between 10 and 15, even more preferably equal to about 14.6.
A weight ratio of less than 15 makes it possible to use a smaller amount of lipid excipient to obtain the same drug effectiveness.
Another aspect of the invention relates to a pharmaceutical composition comprising the adrenaline-containing double layered lipid vesicles as described here.
Said composition can have application in the medical field, in the treatment of cardiac emergencies of various types, such as, for example, cardiac arrest, ventricular fibrillation, pulseless ventricular tachycardia, pulseless cardiac electric activity, cardiac asystole.
The pharmaceutical composition of the invention can be formulated with suitable excipients known to a person skilled in the art. In one embodiment, the composition can be formulated in liquid form, preferably as an injectable solution for parenteral administration, more preferably for intravenous administration.
The adrenaline-containing double layered lipid vesicles for use according to the present invention and the pharmaceutical compositions that comprise them, conceived as described here, are susceptible of numerous modifications and variants, all falling within the scope of the inventive concept; furthermore, all of the details may be replaced by other equivalent elements whose correspondence is known to the person skilled in the art.
Furthermore, it is to be understood that the characteristics of the embodiments described with reference to one aspect of the invention are to be considered valid also in relation to other aspects of the invention, even if not explicitly repeated.
Materials and Methods
1. Materials
Dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine-polyethylene glycol-2000 (DSPE-PEG) were purchased from Lipoid (Ludwigshafen, Germany). The (−) adrenaline, cholesterol, sodium metabisulphite, citric acid monohydrate and disodium hydrogen phosphate dihydrate were purchased from Sigma-Aldrich (Milan, Italy). All the other products used are analytical grade.
2. Preparation of Empty Liposomes with Acidic Internal pH
A buffer solution at pH 2.60, containing citric acid monohydrate (0.094 M) and disodium hydrogen phosphate dihydrate (0.022 M) was prepared first of all, 5 ml of this solution was heated at 65° C. in a temperature-controlled bath. In a separate test tube the lipids: DPPC (27 mg), cholesterol (13 mg) and DSPE-PEG (10.2 mg) were mixed and dissolved in the minimum required amount of chloroform. The lipid solution obtained was poured slowly into the hot aqueous buffer solution under magnetic stirring, which was maintained until complete evaporation of the organic solvent and the formation of multilamellar vesicles of large dimensions. The vesicular dispersion was cooled to room temperature and sonicated with an immersion ultrasound apparatus with a titanium probe, for a total of 120 seconds divided into 5-second sonication cycles alternating with 2-second pauses (Soniprep 150, MSE Crowley, UK). After cooling, the sodium metabisulphite (0.053 M) was added to the liposomal dispersion.
3. Alkalinisation of the External Aqueous Medium and Incorporation of Adrenaline Inside the Vesicles
The aqueous medium containing the liposomal dispersion was titrated with the addition of a 2 M NaOH solution until reaching a pH value of 9.00±0.1, whereas the aqueous environment inside the vesicles maintained an acidic pH value. The solid (−) adrenaline was added to this dispersion until a concentration of 0.6 mg/ml was obtained. The mixture was left overnight at room temperature under constant magnetic stirring to enable the diffusion of the adrenaline into the liposomes. The drug not incorporated in the vesicles, deposited on the bottom of the test tube, was removed from the formulation by centrifugation at 2000 rpm.
4. Physicochemical Characterisation of the Liposomes
The mean diameter and polydispersity index (PDI) of the liposomes were measured by means of the dynamic light scattering (DLS) technique, using a Zetasizer Nano device (Malvern Instruments, UK). The Zeta potential was measured with the same Zetasizer Nano device, with the M3-PALS (Phase Analysis Light Scattering) technique. All of the samples of the liposomal dispersions were analysed 1 hour after preparation or as indicated below.
The X-ray scattering measurements were recorded with an S3-MICRO SWAXS apparatus (HECUS X-ray Systems, Graz, Austria). The Cu Ka radiation with a wavelength of 1.542 A was emitted by a GeniX X-ray generator, which operates at 50 kV and 1 mA. A 1 D-PSD-50 M system (HECUS X-ray Systems, Graz, Austria) with 1024 channels (amplitude 54.0 μiη) was used to measure the X-ray scattering. The operating interval of q (A-1) was 0.003<q<0.600, with q=4πεiη(θ)λ″1, which represents the scattering wave vector. In order to perform the analysis, the liposomal dispersions were loaded into glass capillaries with thin walls (2 mm). The diffraction profiles were recorded for 3 h and analysed by means of GAP (Global Analysis Program) software.
The GAP software enables the SAXS diffractogram of the double layered aggregation structures of the phospholipids (vesicles and lamellar phases) to be interpolated. In particular, the thickness of the membrane (de) is defined as 2(zH+2σH), where zH and σH, obtained from the interpolation of the SAXS curves, respectively represent the distance of the head group of the phospholipid from the centre of the bilayer and the amplitude of the polar head. For the latter parameter, a fixed value of 3 A was considered. The morphology of the liposomes was visualised with Cryo-TEM microscopy, using a JEOL JEM-1230 Electron Microscope (Joel, Japan). The liposomal dispersion was loaded onto a Lacey Formvar/Carbon support, housed on a 300 mesh copper grid; the thin aqueous film was dried with filter paper and immediately placed in liquid ethane, using a Vitrobot apparatus (FEI, Hillsboro, Oreg., USA). The frozen grids were maintained in liquid nitrogen and transferred, again in liquid nitrogen, to a specific support (Gatan, Pleasanton, Calif., USA) at about −180° C. The images were recorded with a CCD camera (Pleasanton, Calif., USA), at a voltage of 100 kV.
5. Encapsulation Efficiency
The liposomal dispersions were purified by ultrafiltration to remove the non-incorporated drug using vertical filtering units with a cutoff of 30 kDa (Amicon Ultra 4, Merck Millipore).
3 ml of the liposomal dispersion was loaded into the upper compartment of the filtering unit and made to rotate at 4000 rpm for two 15-minute cycles, followed by two 30-minute cycles. After each cycle, the filtrate was removed from the lower compartment and an equivalent volume of citrate-phosphate buffer at pH 9 containing sodium metabisulphite (0.053 M) was added into the upper compartment.
The encapsulation efficiency (E %) was calculated by analysing, after the destruction of the vesicles, the concentration of drug present in the purified dispersions and in the non-purified ones by means of high-performance liquid chromatography (HPLC).
6. Drug Release In Vitro
The in vitro studies on the release of adrenaline from the liposomal formulations were conducted with a modified dialysis technique or by means of a “sample and separate” method.
In the former case, a cellulose membrane (Spectra/Por®: cut-off 12-14 kDa MW, pore size 3 nm, Spectrum Laboratories Inc., USA) was positioned between the two compartments of a Franz vertical diffusion cell (Rofarma, Milan). The receptor compartment, with a capacity of 6.5 ml and a diffusion area of 0.636 cm2, was filled with a phosphate buffer solution (PBS pH 7.4) containing sodium metabisulphite (0.053 M), stirred constantly by a small magnetic stirrer at a controlled temperature of 37° C. for the whole duration of the experiment. At the beginning of the
experiment, 0.5 ml of liposomal dispersion and a solution of adrenaline in a citrate-phosphate buffer supplemented with sodium metabisulphite (0.053 M), titrated to pH 9 with NaOH 2 M, were applied on the surface of the membrane as a control.
At 1-hour time intervals, for the 8 hours of the experiment, 1 ml of the solution in the receptor compartment was removed and restored with fresh buffer. The amount of adrenaline contained in the samples removed was analysed by HPLC.
In the “sample and separate” method, 100 μl of liposomes was mixed with 10 ml of release medium in a glass test tube and kept under stirring at 37° C. At regular time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h), 500 μl of the mixture was removed and centrifuged in an ultrafiltration unit (MWCO 10 KDa) to separate the free drug from the drug encapsulated in the liposomes. The amount of adrenaline contained in the samples removed was analysed by HPLC.
7. HPLC Determination of the Adrenaline
The adrenaline contained in the liposomes and in the release medium was determined by fluorescence spectroscopy at an excitation wavelength of 280 nm and emission wavelength of 310 nm, using an Alliance 2690 chromatograph (Waters, Italy). Use was made of an XTerra C18 column (3.5 μiη, 4.6×150 mm, Waters), with a mobile phase composed of 86.5% water, 13.45% acetonitrile and 0.05% acetic acid (v/v) at a flow rate of 0.8 ml/min. A calibration curve was constructed using standard solutions (100-1 ng/μl), having a correlation coefficient (R2) of 0.999.
8. Biocompatibility
The biocompatibility of the formulation was tested on human umbilical vein endothelial cells (HUVECs) obtained from a pool of healthy donors (Promocell, Germany). The cells were cultured in Endothelial Cell Growth Medium 2 (ECGM 2, Promocell) enriched with penicillin (50 units/ml) and streptomycin (50 mg/ml), and maintained in an incubator with a humidified atmosphere, CO2 (5%) at 37° C. The cells were seeded in 96-well plates 24 hours before the treatment with liposomes (density 4×104 cells/well). The adrenaline-containing liposomes or an adrenaline solution (used as a control) were mixed with the culture medium to obtain different concentrations of adrenaline (in the range of 3-120 μg/ml), and the cells were treated with the formulations for 5 hours. At the end of the treatment, the medium was removed and replaced with 100 μl of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS (0.5 mg/ml). After 4 hours of incubation at 37° C., the MTT was removed and 100 μl of dimethyl sulphoxide (DMSO) was added in each well. Absorbance was measured at 570 nm by means of a Synergy 4 plate reader (BioTek, Winooski, Vt., USA). The results are expressed as a percentage of viable cells compared to the untreated cells.
9. Statistical Analysis
The data analysis was performed with R software, version 2.10.1. The results are expressed as the mean±standard deviation. The Tukey test was used to verify any statistical differences between groups, whilst the Student T test was used to compare two samples. Significance was tested at the 0.05 probability level (p).
10. In Vivo Study on Animals
The study was conducted following authorisation of the project (Ref. No. 4263 2/8/2017) and in accordance with national provisions (PD 56/2013, as per Directive 2010/63/EU).
For the pilot study, three animals per group were used. The pig (Landrace/Large White sow, 20 kg, from the Validakis farm, Koropi, Greece) was selected as the model animal. The experimental protocol includes a comparison between 2 groups of animals: group A, receiving commercial adrenaline (adrenaline in an injectable solution 1 mg/ml, Demo, Greece), and group B, receiving a formulation of adrenaline contained in liposomes (experimentally produced according to the present invention), both at a dose of 0.01 mg/Kg.
The measurements, conducted at different time intervals, include haemodynamic recordings: ECG, MAP, CVP, CO, SVR, collection of arterial blood samples for the measurement of pH, pO2, pCO2, base deficit, haemoglobin, lactate and collection of venous blood samples for the measurement of adrenaline and serum biochemistry and plasma metabolomics.
Results
Preparation of the Liposomes
The adrenaline-containing liposomes were prepared exploiting a method of active loading, based on the difference in pH between the aqueous core of the liposomes and the external medium as the driving force for encapsulation. This technique is known in the literature for its effectiveness in loading weak bases or weak acids into liposomes with a higher encapsulation efficiency compared to passive loading methods (for example hydration of the lipid film). In order to induce the encapsulation of a weak base like adrenaline, the pH in the aqueous core of the liposomes must be acidic, whereas the pH of the external phase should be neutral or slightly alkaline. In fact, when adrenaline (pKa 8.6) is incubated at pH 9.00 in the presence of liposomes with an internal pH of 2.60, its neutral form can passively diffuse through the lipid bilayer to reach the aqueous core, where it is trapped in the protonate form, which is no longer able to pass through the membrane in the opposite direction.
One of the main limits of this loading technique is the stability of the drug in the pH range used. Adrenaline oxidises easily at alkaline pH levels, which promote the formation of the toxic derivative adrenochrome, which can be easily recognised because of the reddish brown colour it imparts to aqueous solutions. In order to prevent this phenomenon, the formulation includes an antioxidant, sodium metabisulphite, used at a concentration lower than the maximum concentration accepted by the Food and Drug
Administration (FDA) for parenteral preparations for human use. The
concentration of 10 mg/ml has proven to be sufficient to prevent adrenaline oxidation during the loading process and, together with the protection provided by encapsulation in the vesicles, has made it possible to obtain a long-term stability of the preparation, which shows no colouring for up to 15 months after preparation when stored at room temperature. Physicochemical characterisation of the liposomes
The morphology of the liposomes was initially evaluated by cryo transmission electronic microscopy (cryo-TEM). As may be seen from
The formulation in question was then characterised by SAXS. The diffusion profile (
The size, Zeta potential and polydispersity index (PDI) of the vesicles were measured by Dynamic Light Scattering. The results of these analyses
show that the formulation fundamentally contains liposomes characterised by a low polydispersity index (lower than 0.2), with a mean diameter of 70 nm and a Zeta potential of about −6 mV.
These parameters, together with the pH of the preparation, were monitored for 30 days in order to verify the stability of the system. As may be seen from
Encapsulation Efficiency
The adrenaline-containing liposomes were prepared in lots composed of 3 samples each. In order to verify the repeatability of the production process, the encapsulation efficiency of every sample of each lot was analysed. The mean encapsulation efficiency for each lot was 89±0.5%, 78±2.7%, 87±2.2%. The standard deviation among the samples of the same lot was always lower than 3%, which is considered an acceptable value for a system of this complexity.
In Vitro Release Studies
A necessary introductory note to this section is that there does not exist any standard method approved by a regulatory agency for conducting in vitro studies on the release from nanoparticle-based formulations. All of the methods used are derived in some way from release studies on conventional pharmaceutical forms (for example capsules, tablets, etc. . . . ). The most significant results are the ones capable of establishing an A level in vitro-in vivo (IVIVC) correlation (linear or non-linear point-to-point correlation between absorption in vivo and release in vitro).
In order to best describe the kinetics of the release of adrenaline from the liposomes, the formulations were tested using two different in vitro release methods, and in particular a modified dialysis method using Franz cells and the “sample and separate” method.
In both cases use was made of PBS pH 7.4 at a controlled temperature of 37° C. to simulate the pH, osmolarity and temperature of plasma.
Initially, the modified dialysis method with Franz cells was used.
Adrenaline release from the liposomes was evaluated one day, one week or one month after preparation to identify any differences deriving from destabilisation of the liposome membrane over time.
As may be seen from
The main limit of the modified dialysis method lies in the presence of the dialysis membrane which separates the liposomes from the receiving buffer solution. In fact, Franz cells offer a two-dimensional interface for the diffusion of the free drug from the formulation to the receiving solution. Therefore, although it is an excellent method for evaluating any differences between formulations, we judged this experimental setup to be unsuitable for representing the release that takes place in the circulatory system in vivo.
In contrast, in the “sample and separate” method the vesicles are placed in direct contact (without the interposition of filters or membranes) with the receiving solution, providing an environment that is more similar to circulation in vivo. The results obtained with this method are shown in
Notwithstanding the significant difference in the total amount of adrenaline released, ascribable to the substantial difference between the two methods, both experimental approaches describe a prolonged release by the liposomes when they are exposed to simulated physiological conditions.
In this regard, according to need, the adrenaline release properties of the liposomes can be modified by varying the composition in terms of phospholipids, or including others that can stabilise or destabilise the vesicular structure at the physiological temperature and pH, thus leading respectively to a slower or faster release.
Biocompatibility
Although all the components of the formulation prepared had been individually approved for clinical use in humans by one or more regulatory bodies, and therefore recognised as nontoxic at the concentrations used in the invention, the biocompatibility of the liposomal system described was tested on human umbilical vein endothelial cells (HUVEC). Cells of an endothelial type were selected with the objective of testing the effects of the composition on the tissue that is mostly exposed to it following intravenous injection.
Furthermore, HUVECs are non-cancer cells derived from healthy donors and therefore highly representative of the type of cells that come into contact with the adrenaline-containing liposomes when administered during a cardiac arrest.
The cells were treated with liposomes or with an adrenaline solution, administered at different concentrations. As shown in
In Vivo Study on Animals
The aim of the experimental protocol followed was to study the adrenaline-containing liposome formulation of the present invention in comparison with a commercial formulation of adrenaline commonly used in cardiopulmonary resuscitation. The experiments showed that the liposomes of the present invention do not produce any side effect. The 3 animals that received the liposomal formulation showed an immediate increase in heart rate and in systolic and diastolic pressures, but there was also an observed tendency (though not statistically significant at present) of those pigs to maintain their haemodynamic profile for a longer period of time compared to the animals who received the commercial formulation. This tendency is in agreement with the in vitro release studies, which show a controlled transfer of adrenaline from the liposomes over time, and represents a confirmation of the functioning of the system of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102017000086879 | Jul 2017 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2018/055606 | 7/26/2018 | WO | 00 |
