WO2012059515A1

WO2012059515A1 - Kit for the localized activation of high concentrations of a drug and the procedure thereof

Info

Publication number: WO2012059515A1
Application number: PCT/EP2011/069241
Authority: WO
Inventors: Alberto Gramaglia
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-11-03
Filing date: 2011-11-02
Publication date: 2012-05-10
Anticipated expiration: 2013-05-03
Also published as: ITMI20102043A1; EP2665491A1

Abstract

A kit for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal, comprising liposomal means of encapsulation of the drug, a heater operating within a frequency range suitable for heating said deep anatomical site of interest to a temperature that is at least sufficient to determine the breakdown of said liposomal means as a combined result of the increase in temperature and/or the reduction of the pH brought about by the increase in temperature, and means for vehicling the drug liposome-encapsulated and packaged in a form injectable in said deep anatomical site of interest subjected to hyperthermia where the breakdown of the liposomal means determines the localized release of the drug.

Description

KIT FOR THE LOCALIZED ACTIVATION OF HIGH CONCENTRATIONS OF A DRUG AND THE PROCEDURE THEREOF

DESCRIPTION

The present invention relates to a kit for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal and to a procedure for the localized activation of the drug inhibitor of metalloprotemases in a deep anatomical site of interest.

It is known that tissue remodelling is a complex, multi-phase process that is often involved in physiological states and in pathological states related for example to tumour invasion and to metastases.

The principal groups of enzymes that degrade the extracellular matrix are metalloproteinases (MMPs). They are a family of enzymes involved in tissue remodelling and in morphogenesis.

Many studies have demonstrated that increased activity of metalloproteinases plays a crucial role in various pathological conditions, and it has also been hypothesized that the increased activity of these enzymes can foster the increase in the pathogenesis of certain pathological conditions, such as in the case of cancer.

It is known that the extracellular matrix constitutes the principal barrier to tumour growth and spread, and numerous studies have demonstrated the close association between the expression of various members of the family of metalloproteinases in tumours and their invasive, proliferative and potentially metastatic behaviour.

Many studies have demonstrated that metalloproteinase inhibitors (for example, hydroxamates) represent a family of substances and drugs that have a high capacity for preventing the formation of new tumour vessels and for preventing the metastatic spread of such tumours. Disadvantageously, there is experimental evidence demonstrating that such inhibitors of metalloproteinases reveal high toxicity if administered even at doses slightly below those needed to obtain a substantial oncological effect. In fact, the toxicity of these drugs is principally systemic and particularly affects the joints, which, in the absence of the inhibitors, utilize the metalloproteinases to readjust joint surfaces, whereas in the presence of metalloproteinase inhibitor drugs, ankylosis may affect the joints and result in loss of movement.

The technical task of the present invention is therefore that of supplying a kit for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal and a procedure for the localized activation of the drug inhibitor of metalloproteinases in a deep anatomical site of interest, which obviate the above-described technical drawbacks of the prior art.

In the scope of this technical task, an aim of the invention is to realize a kit capable of locally activating a drug inhibitor of metalloproteinases at high concentrations without, systemic side effects.

The technical task, as well as these and other aims , are achieved according to the present invention through the realization of a kit for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal, characterized in that it comprises liposomal means of encapsulation of the drug, a radio-frequency heater operating within a frequency range suitable for heating said deep anatomical site of interest to a temperature that is at least sufficient to determine the breakdown of said liposomal means as a result of the increase in temperature and/or the reduction of the pH induced by the increase in temperature, and means for vehicling the drug liposome-encapsulated and packaged in a form injectable in said deep anatomical site of interest subjected to hyperthermia where the breakdown of the liposomal means determines the localized release of the drug.

It has been found that the ideal frequency of the heater for deep penetration ranges between 10 and 30 MHz.

Preferably, the liposomal means of encapsulation of the drug is combined with chitosan for its stabilization.

The drug inhibitor of the metalloproteinases preferably comprises hydroxamates or derivatives thereof, or tetracyclines or derivatives thereof.

Preferably, the heating means are capable of increasing the basal temperature of a said anatomical site of interest by an amount ranging between 3 and 8 °C.

Preferably, the kit comprises means for selective cooling of the surface layers of the anatomical site of interest, the means operating in combination with said heating means.

The present invention further provides a drug inhibitor of metalloproteinases characterized in that it has a protective capsule of liposomes, which is susceptible to breakdown resulting from an induced increase in temperature and/or reduction of the pH brought about by the induced increase in temperature in the deep anatomical site of interest in a mammal, wherein the drug is vehicled.

Advantageously, the capsule is combined with chitosan for its stabilization.

The drug inhibitor of metalloproteinases preferably comprises hydroxamates or derivatives thereof, or tetracyclines or derivatives thereof.

It has thus been found that such drug inhibitor of metalloproteinases has a use recommended in the therapeutic treatment of tumours, particularly glioblastomas, owing to its ability to stop and chronicize the tumour. Moreover, the present invention also provides a procedure for the localized activation of high concentrations of a drug inhibitor of metalloproteinases, characterized in that it comprises a step for encapsulation of the drug in liposomes, a step for heating said deep anatomical site of interest to a temperature that is at least sufficient to determine the breakdown of said liposomes as a result of the increase in temperature and/or the reduction of the pH determined by the increase in temperature, and a step for the delivery of the liposome-encapsulated drug in said deep anatomical site of interest subjected to hyperthermia where the breakdown of the liposomes determines the localized release of the drug.

More specifically, the delivery step is preferably carried out systemically or locally.

Preferably, the procedure comprises an additional thermal verification step consisting in positioning an RFID device (acronym of Radio Frequency IDentification) in the drug activation site for the correct definition of the drug supply times at suitable temperatures for the required period of at least 30-60 minutes per procedure.

Further characteristics and advantages of the invention will emerge more clearly from the following description of the invention.

The localized activation procedure of the present invention comprises a first step for the liposomization of molecules of inhibitors of metalloproteinases, such as hydroxamates or the derivatives thereof (for example Batimastat or Marimastat) or tetracyclines or the derivatives thereof, a second step consisting of intravenous or local injection, and a third step consistmg of in situ activation by means of local or loco-regional hyperthermia.

The drug inhibitor of metalloproteinases is liposome-encapsulated by mechanical means or through other known procedures (agitation, ultrasound...). Following this, filtering for purification can be performed, depending upon the specific characteristics of the substance, by means of ultracentrifugation and separation methods, in a manner that ensures the maximum concentration of the liposome- encapsulated drug.

Thereafter, the liposome-encapsulated drug is filtered and rendered injectable. Once it has been injected intravenously or locally, the liposome-encapsulated drug is not immediately active per se, as it is shielded by the liposomal protection that resists without disintegrating under basal body temperatures and pH levels. Its circulation within the vessels thus does not create any systemic toxicity. Advantageously, the liposomal protection can be purposely eliminated by taking advantage of its intrinsic temperature sensitivity. In fact, when the liposomal protection is subjected to a suitable increasing temperature gradient, a disintegrating process begins, releasing and activating the drug. Considering this, the thermal gradient is applied to the specific anatomical area of interest where the disintegration of the liposomal protection is desired and thus the activation of the drug.

A deep anatomical site must be understood as any anatomical site that is located deeper than the integumentary system, as is the central nervous system for example.

In the case of application to a human mammal, in order for it to be effective in the disintegration of the liposomal protection, the process of hyperthermia must bring about an increase in the local temperature of a specific deep anatomical site of interest on the order of about 3-8 degrees, depending upon the typology of the liposomal protection, thus bringing the temperature from 36-37 °C to 41-43 °C. At these temperatures, the senescence of the liposomes occurs at a very fast rate and their disaggregation leads to the release of the drug, which, in turn, acts precisely within the specific area of utilization in which heating was achieved by means of hyperthermia.

In addition to bringing about an increase in local temperature, the hyperthermia generally brings about an acidification of the local interstice, which contributes to facilitating the breakdown of the liposomes and the resulting release of the drug inhibitor of metalloproteinases.

The possibility of activating the aforesaid drugs in the desired areas owing to the synergic effects of the process of localized hyperthermia, allows for the utilization of high drug concentrations.

At high concentrations the drug can thus carry out its anti-angiogenic and anti- metastatic action, without proving to be toxic at the systemic level, in which its activity is negligible, though present at high concentrations, as it is temporarily neutralized by the protection and encapsulation system of the thermosensitive liposomes.

In a preferred embodiment of the invention, the specific drugs utilized are Batimastat or Marimastat.

Some of them are already available in an injectable form per se, that is, contained in a saline solution for injections.

The phospholipids are added to the injectable solution containing Batimastat or Marimastat and then the entire preparation is agitated by ultrasonic or mechanical agitation until achieving optimal formation of the liposomes, which are constituted by phospholipids aggregated into small micelles with a core in which the injectable solution containing Batimastat or Marimastat is found. The liposomes, in particular, are combined with chitosan, which makes it possible to provide the liposomal complex with greater stabilization.

Once the liposome-encapsulated drug has been obtained, it is filtered in order to eliminate the excess unencapsulated product and then injected in the organism preferably intravenously.

The liposome-encapsulated drug is not immediately active per se, as it is shielded by the liposomal protection that resists without disintegrating under basal body temperatures and pH levels of the organism.

In this manner, high concentrations of the drug can be utilized, which can circulate within the vessels without creating any systemic toxicity.

The liposomes have extremely advantageous characteristics, permitting easy disintegration and thus the release of the drug in the specific anatomical sites.

In fact, the liposomes are sensitive to increases in temperature and reductions of the pH.

After having injected the liposome-encapsulated drug, one then proceeds with heating of the anatomical site where the disaggregation of the liposomal protection is desired and thus the activation of the drug as a result.

Hyperthermia is preferably carried out by means of a radio-frequency heater operating within a frequency range of 10 to 30 MHz.

The liposome arrives by means of circulation in the deep anatomical site where the hyperthermia was carried out and it thus encounters a temperature gradient higher than that of the rest of the body and parallelly, a reduction of the pH.

In this manner, a process of disintegration of the liposomal protection begins, leading to the release and activation of drug at high concentrations that can carry out its anti-angiogenic and anti-metastatic action. MATERIALS AND METHODS

LIPOSOMES AND PREPARATION METHOD

The liposomes are phospholipids aggregated into spherules or micelles, small droplets that can contain inside or on the surface substances present in the medium in which the liposome is produced (for example injectable Batimastat, that is, in a saline solution for injections, is added to selected phospholipids so as to permit encapsulation of the greatest amount of the product).

The liposomes can encapsulate various types of biologically active substances:

- water-soluble substances in the aqueous compartments

- liposoluble substances between the double layers of lipids

- amphipathic substances, the lipophilic part of which is found inside the double layer of lipids, and the hydrophilic part protrudes into the aqueous compartment. There are numerous liposome preparation techniques, and the type and dimensions of the liposomes obtained depend upon these techniques:

- mechanical methods (vortex-mixing of dispersions of phospholipids, ultrasound..)

- methods of replacement of organic solvent with water

- methods based on detergent removal (dialysis, chromatographic methods). The liposomization of a drug takes place by adding phospholipids to the injectable solution containing a drug. The entire preparation is then agitated by ultrasonic or mechanical agitation or other means until achieving optimal formation of the liposomes.

The mechanism underlying the formation of the phospholipids is easily explained based on the structural characteristics of the molecules of phospholipids, which, dispersed in water or another solution, spontaneously take on a bilayer arrangement, forming thin sheets that tend to close into vesicular formations with a core containing the solution or water in which the molecules of phospholipids were dispersed previously.

DRUGS AND LIPOSOMIZATION PROCESS

The specific drugs utilized in the present invention are, by way of example, but not limited to, Batimastat or Marimastat.

Among the drugs that can be utilized, there are also various, previously tested, drugs such as doxycycline, prinomastat, etc .

Their functional role is to inhibit the functioning of metalloproteinases by means of various mechanisms, generally blocking the zinc-dependent activation site of the metalloproteinases with domains similar to collagen, virtually tricking the MMP with a false substrate.

Batimastat is already in a form that is injectable per se. The other drugs must be made in a manner proving to make them easily injectable. In fact, while some tetracyclines are already in injectable forms, others are only available in a form for oral administration.

The liposomization procedure does not essentially modify the injectability of a product that is already injectable, but it can render injectable a product that is not injectable per se.

Once the drug takes on an injectable form, the means for delivery of the drug is intravenous delivery, eventually intra-arterial or even local delivery for example by means of ascitic fluid, intralesional injection, etc.

HEATING MEANS AND FUNCTIONING THEREOF.

The methodology utilized to realize the hyperthermia process is that of radio frequency. This methodology subjects the anatomical sector of interest to an electromagnetic field that excites dipole molecules of water until the temperature is brought to desired levels.

More specifically, the radio-frequency generators used (13.56 MHz generators are those used most) also include refrigeration systems in order to prevent patient skin burns (the subcutaneous fat layers retain more heat).

EXAMPLE OF PROCEDURE FOR THE LIPOSOMAL ENCAPSULATION OF DOXYCYCLINE

A solution of DMPC (dimytristoyl-sn-glycero-3-phosphocholine) or a mixture of E-phosphatidylcholine, phosphatidylglycerol and cholesterol in a suitable quantity is added to a round-bottomed flask containing chloroform diluted to the concentration of about 15-25ug\ml Then the drug Doxycycline hydrochloride hemiethanolate hemihydrate is added, which is a lipid-soluble tetracycline with fair MMPI effects, according to the dose one intends to inject (for example 200 mg\ vial).

At this point, with the material being kept in rotation, it undergoes an evaporation process under nitrogen until complete evaporation of the chloroform is achieved (the material can then be kept in a vacuum dryer for no less than 1 hour and a half to eliminate all of the remaining chloroform).

The lipidic-drug layer thus obtained is rehydrated with a TES buffer solution (if needed, preheated to a temperature over, but not well above, the transition temperature- best if around 39-45 °C - so as to increase lipidic phase movement with greater "intake " of the drug in the lipophilic phase of the liposome being formed). Given that the temperature surplus is greater with respect to the transition temperature and the risk of oxidation of the lipids is greater, a temperature of 39 °C is generally used.

This mixture is then subjected to an agitation process at varying degrees of pressure and then to sonication by immersion or in ultrasonic bath until the solution appears perfectly clear without the presence of gross precipitates (aside from the colour of the tetracycline itself).

Then the entire mixture is ultracentrifuged to remove any eventual residue and stored at about 4 °C.

The SUV (small unilamellar vesicles) molecules thus formed generally have a good shape memory.

This illustrative procedure naturally undergoes some changes for non-liposoluble drugs like the one in the example described, while the solutions adopted have an unforeseeable empirical basis and various solutions may be tested to improve the concentration of a drug per SUV.

The possibility of using phospholipids (cholesterol is not a phospholipid, but it is often added in order to stabilize the micellar building blocks) of different types and mixtures is based on the target organ or the drug. The best mixture is empirically determined case by case.

EXAMPLE OF PROCEDURE FOR HYPERTHERMIA OF THE ENCEFALON

(HTRF)

Once the liposome-encapsulated drug has been injected intravenously in the patient, the region where one wants to "open" the liposome is subjected to an electromagnetic field.

The procedure consists in attaching two antennas (or coils or electrodes) in the area of the encephalon, placing them opposite each other so as to include the encephalon area between the two antennas (generally of a size of about 14-18 cm in diameter) and starting emission of the electromagnetic waves, raising the power until reaching a measurable skin temperature of at least 39 °C-40 °C or according to internal measurements at the bottom of the external auditory canal filled with saline solution, registering temperatures there on the order of 41 °C-43 °C, or by means of an RFID device (acronym of Radio Frequency IDentification) inserted transcutaneously and/or intraoperatively and/or intracavitarily inside the brain case in the tumour or in the immediate vicinity of the anatomical area.

The thermal treatment continues for about 1 hour, reaching the maximum temperature foreseen internally of about 41-42.5 °C usually after several minutes (15 max.). The treatment is repeated many times within the span of the half-life of the liposome-encapsulated drug, attempting, when possible, to set intervals of at least 24 hours between one administration and another up to 6-9 sessions (this procedure can be avoided in the case of a short half- life and thus carrying out the treatments daily for 4-5 sessions).

The RPID device makes it possible to correctly define the delivery times of treatment at suitable temperatures, particularly for the required time of at least 30- 60 minutes per procedure.

The hyperthermia device is in particular a radio-frequency device (13.56 MHz) and it is equipped with systems of cutaneous cooling by means of plates (coils) inside bags cooled with an external refrigeration unit and controlled by a generator capable of supplying power of about 1200 W.

For thermal treatment of the encephalon, HTRF therapy can begin even slightly before the injection itself, preceding the administration of the drug with an injection of steroids so as to raise the local pH.

In the case of drugs having a potential daily dose (doxycycline), the therapy can always be combined with an additional administration by injection; however, in the case of drugs like hydroxamates (some of which having very long half-life periods), the total dose can be administered in a lower number of times.

Claims

1. A kit for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal, characterized in that it comprises liposomal means of encapsulation of the drug, a radio-frequency heater operating within a frequency range suitable for heating said deep anatomical site of interest to a temperature that is at least sufficient to determine the breakdown of said liposomal means as a result of the increase in temperature and/or the reduction of the pH induced by the increase in temperature, and means for vehicling the drug liposome- encapsulated and packaged in a form injectable in said deep anatomical site of interest subjected to hyperthermia where the breakdown of the said liposomal means determines the localized release of the drug.

2. The kit for the localized activation of high concentrations of a dmg inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal according to the preceding claim, characterized in that said frequency range is between 10 and 30 MHz.

3. The kit for the localized activation of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal according to any one of the preceding claims, characterized in that said liposomal means is combined with chitosan for its stabilization.

4. The kit for the localized activation of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal according to any one of the preceding claims, characterized in that said heater is programmed so as to supply the thermal power needed for an increase within a range of 3 to 8 °C in the temperature of said deep anatomical site.

5. The kit for the localized activation of a drug inhibitor of metalloprotemases in a deep anatomical site of interest in a mammal according to any one of the preceding claims, characterized in that it comprises means for selective cooling of the surface layers of the deep anatomical site of interest, the means operating in combination with said heater.

6. The kit for the localized activation of a drug inhibitor of metalloprotemases in a deep anatomical site of interest in a mammal according to any one of the preceding claims, characterized in that said drug inhibitor of the metalloprotemases comprises hydroxamates or derivatives thereof.

7. The kit for the localized activation of a drug inhibitor of metalloprotemases in a deep anatomical site of interest in a mammal according to any one of the preceding claims, characterized in that said drug inhibitor of the metalloprotemases comprises tetracyclines or derivatives thereof.

8. A drug inhibitor of metalloprotemases characterized in that it has a protective capsule of liposomes, which is susceptible to breakdown resulting from the increase in temperature and/or the reduction of the pH induced by the increase in temperature in the deep anatomical site of interest in a mammal, wherein said drug is vehicled, said capsule being combined with chitosan for its stabilization.

9. The drug inhibitor of metalloprotemases according to claim 8, characterized in that it comprises hydroxamates or derivatives thereof.

10. The drug inhibitor of metalloprotemases according to claim 8, characterized in that it comprises tetracyclines or derivatives thereof.

1 1. A use of a drug in accordance with any one of claims 8 to 10 in order to hinder the growth of a glioblastoma.

12. A procedure for the localized activation of high concentrations of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in a mammal, characterized in that it comprises a step for encapsulation of the drug in liposomes, a step for stabilization of the liposomes with chitosan, a step for heating said deep anatomical site of interest to a temperature that is at least sufficient to determine the breakdown of said liposomes as a result of the increase in temperature and/or the reduction of the pH induced by the increase in temperature, and a step for the delivery of the liposome-encapsulated drug in said deep anatomical site of interest subjected to hyperthermia where the breakdown of the liposomes determines the localized release of the drug.

13. The procedure for the localized activation of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in an animal according to the preceding claim, characterized in that said delivery step is earned out systemically.

14. The procedure for the localized activation of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in an animal according to claim 12, characterized in that said delivery step is carried out locally.

15. The procedure for the localized activation of a drug inhibitor of metalloproteinases in a deep anatomical site of interest in an animal according to claim 12, characterized in that it comprises an additional thermal verification step consisting in positioning an RPID device in the drug activation site for the correct definition of the drug supply time at a suitable temperature.