MX9706402A

Stabilized gas emulsion containing phospholipid for ultrasound contrast enhancement.

Abstract

A gas emulsion forming composition comprising a dry, hollow, particulate, approximately microspherical material permeated with a gas or gas mixture, which upon dissolution in aqueous liquid forms a gas emulsion comprising a plurality of bubbles surrounded by a layer of at least a first and a second surfactant, wherein the first surfactant consists essentially of a phospholipid or mixture of phospholipids having at least one acyl chain which comprises at least 10 carbon atoms, and comprising at least about 5 % w/w of total surfactant, and wherein the second surfactant may or may not be a phospholipid and is more water soluble than the first surfactant; kits for preparing such microbubbles; and methods for using such microbubbles as contrast agents.

MX9706402A, drawing sheet 1 — Sheet 1 of 3

Term

Term ended

Expired 21 August 2017, 9.1 years ago.

43 claims: 5 independent, 38 dependent

1
REIVINDICACIONES 1. Un método para formar una emulsión de gas, este método comprende las etapa de:suministrar un recipiente, que tenga dentro de él un material seco, hueco, particulado, aproximadamente microesférico, el cual comprende cuando menos un primero y un segundo agentes tensoactivos, un monómero o polímero hidrofílico o sus combinaciones, y un gas o una mezcla de gases, este primer agente tensoactivo incluye un fosfolipido o una mezcla de fosfolipidos, que tienen cuando menos una cadena de acilo, la cual comprende al menos 10 átomos de carbono, y comprende cuando menos un 5% en peso/peso del agente tensoactivo total, el segundo agente tensoactivo es más soluble en agua que el primer agente tensoactivo;agregar un liquido acuoso al recipiente;disolver substancialmente el material microesférico en el liquido acuoso, formando asi una emulsión de gas dentro del recipiente, esta emulsión de gas comprende burbujas del gas o la mezcla de gases rodeadas por una capa del primero y el segundo agentes tensoactivos.
2
El método de la reivindicación 1, en que el segundo agente tensoactivo comprende un ácido graso, una sal de un ácido graso, un éster de azúcar de uno o más ácidos grasos, un copolimero de polioxipropileno y polioxietileno, -9191 un alquilglucósido no iónico, un polisorbato o una combinación de los mismos.
3
El método de la reivindicación 1, en que el segundo agente tensoactivo comprende un fosfolipido, una mezcla de fosfolípidos, una fosfocolina o un lisofosfolipido donde cadena de acilo comprende no más de 14 átomos de carbono.
4
El método de la reivindicación 1, en que el primer agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena incluye de 12 a 18 átomos de carbono, y el segundo agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, al menos una cadena comprende de 6 a 12 átomos de carbono.
5
El método de la reivindicación 1, en que el monómero o polímero hidrofilico o una combinación de los mismos, comprenden un almidón o un almidón derivado.
6
El método de la reivindicación 1, en que el gas o la mezcla de gases comprende un fluorocarbono o una mezcla de fluorocarbonos.
7
El método de la reivindicación 1, en que el gas o la mezcla de gases comprende un primer gas no de fluorocarbono en mezcla con un segundo gas, este segundo gas tiene -9292 una presión de vapor menor de 7 60 mm de Hg a una temperatura de 37°C.
8
El método de la reivindicación 7 en que el primer gas consiste esencialmente de nitrógeno o de aire, y el segundo gas consiste esencialmente del perfluorohexano.
9
Un método para formar una composición precursora de una emulsión de gas, este método comprende las etapas de:dispersar una solución acuosa, que incluye un monómero o polímero hidrofílico, o una combinación de los mismos, y un primero y segundo agentes tensoactivos, en que el primer agente tensoactivo consiste esencialmente de un fosfolipido o una mezcla de fosfolípidos, que tiene cuando menos una cadena de acilo, la cual incluye cuando menos 10 átomos de carbono, y comprende cuando menos alrededor del 5% en peso/peso del agente tensoactivo total, y el segundo agente tensoactivo es más soluble en agua que el primer agente tensoactivo;y secar por rociado la dispersión, para crear un material seco, hueco, particulado, aproximadamente microesférico, el cual, cuando se combina con un medio acuoso, forma una emulsión de gas ecogénica, que incluye burbujas de un gas rodeadas por una capa del primero y segundo agentes tensoactivos. -9393
10
El método de la reivindicación 9, que además comprende la etapa de encerrar el material microesférico en un recipiente, con un gas o una mezcla de gases, en que este gas o mezcla de gases comprende un fluorocarbono o una mezcla de fluorocarbonos.
11
El método de la reivindicación 9, en que el monómero o polímero hidrofílico comprende aproximadamente del 1 al 99% en peso/peso del material seco, microesférico, y el agente tensoactivo total incluye aproximadamente del 0.05 al 90% en peso/peso del material seco, microesférico. agente de inflado comprende una substancia seleccionada del grupo que consta del cloruro de metileno, el Freon 113, el perfluorohexano y el bióxido de carbono.
12
14. El método de la reivindicación 9, en que el segundo agente tensoactivo comprende un ácido graso, una sal de un ácido graso, un éster de azúcar de uno o más ácidos grasos, un copolímero de polioxipropileno y polioxietileno, un alquilglucósido no iónico, un polisorbato, o una combinación de los mismos. -9494
13
15. El método de la reivindicación 9, en que el segundo agente tensoactivo comprende un fosfolipido, una mezcla de fosfolipidos, una fosfocolina, o un lisofosfolipido, donde cada cadena de acilo incluye no más de 14 átomos de carbono.
14
16. El método de la reivindicación 9, en que el primer agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena incluye de 12 a 18 átomos de carbono, y el segundo agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena incluye de 6 a 12 átomos de carbono.
15
17. El método de la reivindicación 9, en que el monómero o polímero hidrofílico, o una combinación de los mismos, comprende un almidón o un almidón derivado.
16
18. El método de la reivindicación 10, en que el gas o la mezcla de gases comprende un primer gas no de fluorocarbono en mezcla con un segundo gas, este segundo gas tiene una presión de vapor de menos de 7 60 mm de Hg a una temperatura de 37°C.
17
19. El método de la reivindicación 18, en que el primer gas consiste esencialmente del nitrógeno o el aire, y el segundo gas consiste esencialmente del perfluorohexano. -9595
18
20. Una composición que forma una emulsión de gas, esta composición comprende:un recipiente;un material seco, hueco, particulado, aproximadamente microesférico, dentro del recipiente, que incluye cuando menos un primero y un segundo agente tensoactivo, y una cantidad suficiente de un monómero o polímero hidrofilico o una combinación de los mismos, para impartir integridad estructural al material microesférico;en que el primer agente tensoactivo consiste esencialmente de un fosfolípido o una mezcla de fosfolípidos, que tiene cuando menos una cadena de acilo, la cual comprende al menos 10 átomos de carbono, y que incluye cuando menos un 5% en peso/peso del agente tensoactivo total, y en que el segundo agente tensoactivo es más soluble en agua que el primer agente tensoactivo;y un gas o una mezcla de gases dentro del recipiente, impregnados dentro del material microesférico.
19
21. La composición de la reivindicación 20, en que el segundo agente tensoactivo comprende un ácido graso, una sal de un ácido graso, un éster de azúcar de uno o más ácidos grasos, un copolímero de polioxipropileno y polioxietileno, un alquilglucósido no iónico, un polisorbato, o una combinación de los mismos. -9696
20
22. La composición de la reivindicación 20, en que el segundo agente tensoactivo comprende un fosfolipido, una mezcla de fosfolipidos, una fosfocolina o un lisofosfolípido, donde cada cadena de acilo incluye no más de 14 átomos de carbono.
21
23. La composición de la reivindicación 20, en que el primer agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena comprende de 12 a 18 átomos de carbono, y el segundo agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena comprende de 6 a 12 flúorocarbono en mezcla con un segundo gas, este segundo gas tiene una presión de vapor menor de 7 60 mm de Hg, a una temperatura de 37°C.
22
26. La composición de la reivindicación 25, en que el primer gas consiste esencialmente de nitrógeno o de aire, y el segundo gas consiste esencialmente del perfluorohexano.
23
27. La composición de la reivindicación 20, en que el monómero o polímero hidrofilico comprende un almidón o un almidón derivado.
24
28. Una medio de contraste de ultrasonido de emulsión de gas, este medio comprende:burbujas de gas o de una mezcla de gases, que comprende un fluorocarbono o una mezcla de fluorocarbonos, que tienen cuando menos cuatro átomos de carbono rodeados por una capa de agente tensoactivo, que incluye al menos un primero y un segundo agentes tensoactivos, este primer agente tensoactivo consiste esencialmente de un fosfolipido o una mezcla de fosfolípidos, que tiene cuando menos una cadena de acilo, la cual comprende al menos 10 átomos de carbono, y que incluye al menos un 5% en peso/peso del agente tensoactivo total, y en que el segundo agente tensoactivo es más soluble en agua que el primer agente tensoactivo.
25
29. El medio de contraste de la reivindicación 28, en que el segundo agente tensoactivo comprende un ácido graso, una sal de n ácido graso, un éster de azúcar de uno o más ácidos grasos, un copolímero de polioxipropileno y polioxietileno, un alquilglucósido no iónico, un polisorbato, o una combinación de los mismos.
26
30. El medio de contraste de la reivindicación 28, en que el segundo agente tensoactivo comprende un fosfo -9898 lipido, una mezcla de fosfolípidos, una fosfocolina o un lisofosfolípido, donde cada cadena de acilo incluye no más de 14 átomos de carbono.
27
31. El medio de contraste de la reivindicación 28, en que el primer agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, al menos una cadena incluye de 12 a 18 átomos de carbono, y el segundo agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, cuando menos una cadena incluye de 6 a 12 átomos de carbono.
28
32. El medio de contraste de la reivindicación 28, en que el gas o la mezcla de gases comprenden un primer gas no de fluorocarbono en mezcla con un segundo gas, este segundo gas tiene una presión de vapor menor de 760 mm de Hg, a una temperatura de 37°C.
29
33. El medio de contraste de la reivindicación 32, en que el primer gas consiste esencialmente del nitrógeno o el aire, y el segundo gas consiste esencialmente del perfluorohexano.
30
34. Una composición que forma una emulsión de gas, esta composición comprende:un recipiente;una dispersión, secada por rociado, de un monómero o polímero hidrofilíco o una combinación de los mismos, y un -9999 primero y segundo agentes tensoactivos, en que el primer agente tensoactivo consiste esencialmente de un fosfolipido o una mezcla de fosfolipidos, que tiene cuando menos una cadena de acilo, la cual comprende al menos 10 átomos de carbono e 5 incluye cuando menos un 5% en peso/peso del agente tensoactivo total, el segundo agente tensoactivo es más soluble en agua que el primer agente tensoactivo;y un gas o una mezcla de gases dentro del recipiente, impregnado dentro de la dispersión secada por rociado. 10
31
35. La composición de la reivindicación 34, en que el segundo agente tensoactivo comprende un ácido graso, una sal de un ácido graso, un éster de azúcar de uno o más ácidos grasos, un copolímero de polioxipropileno y polioxietileno, un alquilglucósido no iónico, un polisorbato, o una combináis ción de los mismos.
32
36. La composición de la reivindicación 34, en que el segundo agente tensoactivo comprende un fosfolipido, una mezcla de fosfolipidos, una fosfocolina o un lisofosfolipido, donde cada cadena de acilo comprende no más de 14 átomos de 20 carbono.
33
37. La composición de la reivindicación 34, en que el primer agente tensoactivo comprende una fosfatidilcolina con una o más cadenas de acilo, al menos una cadena comprende de 12 a 18 átomos de carbono, y el segundo agente tensoactivo -100100 comprende una fosfatidilcolina, con una o más cadenas de acilo, cuando menos una cadena comprende de 6 a 12 átomos de carbono.
34
38. La composición de la reivindicación 34, en que el gas o la mezcla de gases comprende un fluorocarbono o una mezcla de fluorocarbonos.
35
39. La composición de la reivindicación 34, en que el gas o la mezcla de gases comprende un primer gas no de fluorocarbono, en mezcla con un segundo gas, este segundo gas tiene una presión de vapor menor de 7 60 mm de Hg a una temperatura de 37°C.
36
40. La composición de la reivindicación 39, en que el primer gas consiste esencialmente de nitrógeno o de aire, y el segundo gas consiste esencialmente del perfluorohexano. agente de inflado comprende una substancia seleccionada del grupo que consta del cloruro de metileno, el· Freon 113, el perfluorohexano y el bióxido de carbono.
37
43. Un método para obtener un medio de contraste de ultrasonido, que comprende disolver substancialmente la composición de la reivindicación 34 en un líquido acuoso, -101101 formando así una emulsión de gas dentro del recipiente, esta emulsión de gas incluye burbujas que contienen el gas o la mezcla de gases rodeado por una capa del primero y segundo agentes tensoactivos.
38
44. Un método para obtener un medio de contraste de ultrasonido, que comprende disolver substancialmente la composición de la reivindicación 40 en un líquido acuoso, que forma así una emulsión de gas dentro del recipiente, esta emulsión de gas incluye burbujas que contienen al menos el nitrógeno o el aire y el perfluorohexano, rodeado por una capa del primero y segundo agentes tensoactivos.
39
45. Un método para formar imágenes de un objeto o cuerpo, este método comprende las etapas de:disolver substancialmente la composición que forma la emulsión de gas, según la reivindicación 20, en un líquido acuoso, para producir una emulsión de gas;introducir la emulsión de gas dentro del objeto o cuerpo;y formar la imagen ultrasónicamente de al menos una porción del objeto o cuerpo.
40
4 6. Un método para formar imágenes de un objeto o cuerpo, este método comprende las etapas de:introducir el medio de contraste de ultrasonido, según la reivindicación 28, dentro del objeto o cuerpo;y -102102 formar la imagen ultrasónicamente de al menos una porción del objeto o cuerpo.
41
47. Un método para formar imágenes de un objeto o cuerpo, este método comprende las etapas de:introducir el· medio de contraste de ultrasonido, según la reivindicación 33, dentro del objeto o cuerpo;y formar la imagen ultrasónicamente de al menos una porción del objeto o cuerpo.
42
48. Un método para formar imágenes de un objeto o cuerpo, este método comprende las etapas de:disolver substancialmente la composición que forma la emulsión de gas, según la reivindicación 34, en un líquido acuoso, para producir una emulsión de gas;introducir la emulsión de gas dentro del objeto o cuerpo;y formar la imagen ultrasónicamente de al menos una porción del objeto o cuerpo.
43
4 9. Un método para formar imágenes de un obj eto o cuerpo, este método comprende las etapas de:introducir el medio de contraste de ultrasonido, según la reivindicación 44, dentro del objeto o cuerpo;y formar la imagen ultrasónicamente de al menos una porción del objeto o cuerpo. -103- F/a 7 F/a 2 -1042/2 F/σ. 3 -105-

Independent claims43

513 paragraphs in 4 sections, as filed

WQRLD INTELLECTUAL PROPERTY ORGANIZED BY TION TflAF lnternational Burea »-

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

<td>(51) fatmaÜMial Patent Clnariflcatlon 6: Α61Κ49ΛΧ)</td><td>To the</td><td colspan="2">(11) International Pubücatton Number: WO 94/26746 (43) lnternational Publication Date: 6 September 1996 (06.09.96)</td>

<td colspan="3">(21) InteraatiMal AppUcation Number: PCI7US96 / 01922 (22) brteraatkmnl Fttlng Date: February 15, 1996 (02/15/96) (30) Priority Data: 08 / 395,680 28 February 1995 (28.02.95) US (71) Applicant: ALLIANCE PHARMACEUTICAL CORP. (US / US); 3040 Science Partí Roed. San Diego, CA 92121 (US). (72) Inventor *: TREVINO, Leo, Apartment 5210,7665 Palmilla Drive, San Diego, CA 92122 (US). SCHUTT, Emeat, Gcorge; 12139 Ragweed Sorel, San Diego, CA 92129 (US). KLEIN, David, H „4615 Bucklngham Lañe. Carlibed, CA 92008 (US). TARARA, Thomas, E .; 3072 A Street, San Diego, CA 92102 (US). WEERS, Jeffiy. G .; 12191 Salix Way, San Diego, CA 92129 (US). KABALNOV, Alexey; 12604 Torrey Bluff Drive, # 393. San Diego, CA 92130 (US). (74) Agent: ALTMAN, Daniel, E .; Knobbe. Manen *. Olson and Bear, Suite 1600, 620 Newport Cerner Drive. Newpcrt Beach, CA 92660 (US).</td><td>(81) Derignated Statec AL. A.M. AT, AU, AZ, BB, BG. BR, BY. CA, CH, CN, CZ, DE, DK, EE, ES. FI, GB. GE, HU, IS. JP, KE, KG, KP, KR. KZ, LK, LR, LS, LT, LU. LV, MD, MG, MK. MN. MW, MX, NO, NZ, PL, PT, RO. RU, SD, SE, SG, SI, SK, TJ, TM. TR. ΤΓ, UA, UG. UZ. VN, ARITO patent (KE, LS, MW, SD, SZ, UG), Eurasian patent (AZ, BY, KG, KZ, RU. TJ, TM), European patent (AT. BE. CH, DE, DK, ES , FR, GB, GR, IB, LU. LU, MC, NL, PT. SE). OAPI patent (BF, BJ, CF. CU. CI. CM, GA. GN, ML. MR, NE, SN. TD, TG). Publlabed With international eearch repon. Befare the cxpiration of the tinte limit for am ending the ciaims and lo be republiehed in the event of the receipt of amendmentí.</td>

<td colspan="4">(54) Tille: STABIL1ZED GAS EMULSION CONTAINING PHOSPHOLIPID FOR ULTRASOUND CONTOAST ENHANCEMENT (57) Abstract A gas emulsion forming compoaition compression to dry, bollow. paniculate, approximately micnMpherieal material permeated with a ge or gas mixture, which upon dlwolution in aquecua liquid forma a ga> emulsion compriaíng a plunlity of bubblet lumounded by a layer of at least * finí and a second lurfactant, wheretn the fltat aurfactant consist esmntially of a phoapbolipid or mixtun of phospholipids having at teaat one acyl chain which compresses at least 10 carbon atonta, and ccmpriting at leaat about 5% w / w of total surfactant. and wheretn the second wrfactant may or may not be a phospholipid and is more water soluble than the finí surfactent; kit * for prtpiring such microbubblcs, and methods for uaing such microbubbles as hire agenta.</td>

-1 STABILIZED GAS EMULSION. WHAT CONTAINS PHOSPHOLIPIDS

TO INCREASE THE CONTRAST OF THE ULTRASOUND

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention includes a method for preparing long-lived, stable gas emulsions for increasing ultrasound contrast and other uses, and for the gas emulsion compositions thus prepared.

<td> 10</td><td colspan="4">Background of the Technique</td>

<td>The</td><td>technology</td><td>of the</td><td>ultrasound</td><td>supplies a</td>

<td>alternative</td><td>important and</td><td>plus</td><td>economic to</td><td>the techniques of</td>

<td>formation of</td><td>images that</td><td>íjsan</td><td>the radiation</td><td>ionic. While</td>

Numerous conventional imaging technologies are available, for example, magnetic resonance imaging (MFI), computed tomography (CT), and positron emission tomography (PET), each of these techniques using extremely equipment expensive. Likewise, CT and PET use ionic radiation. Unlike these techniques, ultrasound imaging equipment is relatively inexpensive. Also, ultrasound imaging does not use ionizing radiation.

Ultrasound imaging makes use of differences in tissue density and composition,

-2 which affect the reflection of sound waves by these tissues. The images are especially sharp where there are different variations in tissue density or compressibility, such as at the tissue interface. These interfacial zones between solid tissues, the skeletal system and various organs and / or tumors, easily form images by ultrasound.

Therefore, in many imaging applications, ultrasound performs adequately without using contrast-enhancing agents; however, for other applications, such as visualization of flowing blood, efforts have been made to develop such agents to deliver contrast enhancement. A particularly significant application for such contrast agents is in the area of perfusion imaging. Contrast agents for ultrasound can improve imaging of the blood flowing in the heart muscle, kidneys, liver, and other tissues. This, in turn, will facilitate diagnostic research, surgery, and therapy related to the image tissues. A contrast agent for a pool of blood will also allow imaging based on blood content (eg, in tumors and inflamed tissues) and will aid in visualization of the fetus, increasing only maternal circulation.

A variety of ultrasound contrast enhancing agents have been proposed.

The most successful agents generally consist of dispersions or small gas bubbles that can be injected intravenously. More typically, the bubbles are injected into the blood stream of a living body from which the image is to be formed.

The bubbles then supply a physical object in the flowing blood, that is, of a different density and much greater compressibility than the fluid tissue and blood that surround it. As a result, these bubbles can easily form images with ultrasound. To cross the blood vessels, the bubbles must be less than 10 μτη in diameter and have been named microbubbles. These microbubbles can form in a liquid in a variety of different ways. Simple examples are vigorous stirring or forcing a gas into a liquid through a small hole. In the absence of additional ingredients, the gas will be in direct contact with the condensed medium (i.e., bubbles, however, such bubbles tend to shrink rapidly, due to the diffusion of the gas trapped in the surrounding liquid. Furthermore, pure microbubbles, as shown, produce adverse responses, such as complement activation (see, eg, KA Shastri et al. (1981) Undersea Bioraed. Res., 18, 157). Attempts to prolong the life of microbubbles to increase their usefulness have focused on the addition of stabilizing agents which can trap gas bubbles, slowing the diffusion of gas into the surrounding liquid.

Most microbubble compositions have failed to deliver contrast enhancement that lasts even a few seconds, let alone minutes. This greatly limits its usefulness. Microbubbles, therefore, have been constructed in various ways in an attempt to increase their effective life by increasing contrast. Various routes have been attempted, such as the use of gelatins or albumin microspheres, which initially form in the liquid suspension, and which trap gas during solidification. However, the solid phase covers that encapsulate gases have generally proven to be too brittle or too gas permeable to have a satisfactory life in vivo. Also, thick covers (eg, albumin, sugar, or other viscous materials) reduce the compressibility of the bubbles, thus reducing their echogenicity for the short time that they may exist. Solid particles or liquid emulsion droplets that produce gas or boil when injected (as in Quay's PCT / US94 / 00422) pose the danger of supersaturating blood with gas or steam. This would lead to the formation of a small number of large bubbles that would produce strokes at the few available nucleation sites, rather than the large number of small bubbles attempted. Furthermore, the bubbles created in vivo, in this way, will be pure and, consequently, will have the complement activation problem, described above.

The use of surfactants as stabilizers for gas bubble dispersions has also been explored. Surfactants are materials that tend to form an inter-facial layer on the interface of the polar substance with a non-polar substance. Its active surface behavior comes from the existence of both a hydrophilic region (which often comprises an end which is usually named as the head), which tends to associate with the polar substance, as a hydrophobic region (which often includes the other end, which is usually named as the tail), which tends to associate with the nonpolar substance. When stabilized, the interfacial layer affects the characteristics of the polar / nonpolar interface. When surfactants are present, the gas can be separated from the liquid by a layer

-6 interface, which can comprise a wide variety of surfactant materials.

Some contrast-enhancing agents, which contain the surfactant, trap gas bubbles in another way, for example, in an aqueous liposome nucleus. These liposomes are more or less spherical pockets comprised of an aqueous nucleus bounded by one or more closed, concentric layers of bimolecular phospholipid. These phospholipids, which are natural components of cell membranes, are also well known for their surfactant properties. In U.S. Patent No. 5,334,381 to Unger, gas bubble-containing liposomes are created by several different mechanisms. Likewise, US Patent No. 4,900,540 to Ryan et al. , reveals phospholipid liposomes that contain a gas or a gas precursor. Presumably, gas bubbles trapped within the liposomes slowly escape outside, thereby increasing the effectiveness of the contrast agent. It can be noted that this use of a surfactant does not imply the presence of an interface layer of surfactant at the gas / liquid interface. Rather, small gas bubbles are trapped in a larger volume of aqueous liquid, which itself binds to the uni- or multi-lamellar liposomal structure.

Contrast agents containing surfactant materials can use liposomes in other ways. For example, in the patents of E. 0. A., Nos. 5,380,519 and 5,271,928 of Schneider et al., Microbubbles prepared from freeze-dried liposomes are described. According to this description, reconstitution in water of the dry powdery formulation, created by lyophilization, of a suspension of liposomes, creates a dispersion of gas bubbles in suspension, with the liposomes filled with water. The microbubbles, thus prepared, are indicated to be surrounded by a rather evanescent envelope of the surfactant. Although such an evanescent surfactant layer will generally be expected to have no persistence, and that these microbubbles will therefore not be stable for an extended period of time, Schneider et al. Theorize that the surfactant laminated to or from neighboring liposomes filled with water, it stabilizes the gas present in the system in the form of microbubbles.

It will be readily appreciated that a liposome-dependent contrast enhancing agent requires prior formation of the liposomes, and therefore limits the major component of the stabilizing surfactant to a type which is capable of forming liposomes. Likewise, liposome preparation involves sophisticated and time-consuming manufacturing.

Even in the presence of stabilizing compounds or structures, the trapped gases are under increased pressure in the bubble due to the surface tension of the surrounding surfactant, as described by the Laplace equation (ΔΡ = 2y / r). This increased pressure rather facilitates shrinkage and disappearance of the bubble as the gas moves from a high pressure area (in the bubble) to a lower pressure environment (in any surrounding liquid, which is not saturated with gas at this high pressure, or within a bubble of larger diameter, less pressure).

One purpose for dealing with these problems is outlined in Quay's patent PCT / US92 / 07250. Quay bubbles using selected gases, which are gaseous at body temperature (37 ° C) and have reduced solubility in water, high density, and reduced diffusivity of gas in solution, compared to air. Although reduced water solubility and diffusivity can affect the rate at which the gas leaves the bubble, numerous problems exist with Quay bubbles. Bubble formation of sufficiently small diameter (eg 3-5 pm) requires a high input of energy. This is a disadvantage in that sophisticated bubble preparation systems must be supplied at the site of use. Likewise, the Quay criteria for gas selection are incorrect in that they fail to consider certain major causes of bubble shrink, that is, the effects of surface tension on bubbles, the effects of gas surfactants and osmotics. , and these errors result in the inclusion of undesirable gases and the exclusion of certain optimally suitable gases.

Accordingly, there is a need in the art for compositions, and a method of preparing these compositions, that supply, or utilize, a long-lived, contrast enhancing agent that is biocompatible, readily prepared, and delivers a contrast enhancement. Superior in ultrasound imaging.

Compendium of the Invention

In accordance with the present invention, a gas emulsion ultrasound contrast enhancing means is provided, incorporating a mixture of surfactants as bubble stabilizing agents. At least one of such surfactants is a hydrophobic phospholipid or a mixture of phospholipids. At least one second surfactant is supplied, which may or may not also be

-1010 a phospholipid or a mixture of phospholipids, but which is more hydrophilic than the phospholipid or the combination of phospholipids provided as the first surfactant. Such a phospholipid stabilized gas emulsion has a long longevity in vivo.

In one embodiment of the present invention, a gas emulsion composition is prepared by first dispersing, in an aqueous solution, a hydrophilic monomer or polymer or combinations thereof, a first and a second surfactant and an inflation agent. The first surfactant is a phospholipid or a phospholipid mixture having at least one acyl chain comprising at least 10 carbon atoms and including at least 5% w / w of the total surfactant, and the second surfactant is more soluble in water than the first surfactant.

The dispersion is then spray dried to evaporate the inflation agent and create a rough, particulate, hollow, dry material. This dry particulate material is exposed to at least a first gas and can then be dissolved in an aqueous liquid, thus forming an aqueous gas emulsion composition, where this composition comprises gas bubbles surrounded by a layer of the first

-1111 and second surface active agents, whose stability is independent of liposomes.

The second surfactant can be comprised of a wide variety of materials. Some specific examples include fatty acids, fatty acid salts, fatty acid sugar esters, polyoxypropylene-polyoxyethylene copolymers, nonionic alkyl glucosides, and polysorbates. Especially suitable gas emulsions are prepared when the second surfactant comprises a phospholipid or a mixture of phospholipids having one or more acyl chains, in which each acyl chain comprises no more than 14 carbon atoms. The hydrophilic monomer or polymer, or combinations thereof, can be a starch.

The gas, or combination of gases, that permeate the dry particulate material may also be selected from a wide variety of substances, including air, nitrogen, carbon dioxide, or other gases normally present in the blood, and may also be selected from a wide variety of substances, including air, nitrogen, carbon dioxide, or other gases normally present in the blood, and can also be an organic material, such as a fluorocarbon. Preferably one of the supplied gases has a vapor pressure of less than 760

-1212 mm Hg at 37 ° C. A particularly preferred embodiment uses perfluorohexane-saturated nitrogen.

The present invention also includes containers for gas emulsion-forming compositions of dry gas permeated particulate material, and methods for imaging an object or body part or body cavity, by introducing an emulsion composition. of gas that contains a phospholipid within the object or part of the body or body cavity, and forms the image of at least a portion of the body by ultrasound.

Brief Description of the Drawings

Figure 1 is a perspective view of a two-chamber bottle containing a microbubble-forming preparation, with an aqueous solution in an upper chamber and solid and gaseous ingredients in the lower chamber.

Figure 2 illustrates the bottle of Figure 1, where the aqueous solution has been mixed with the solid ingredients to form microbubbles for administration to a patient.

Figure 3 is a perspective view of an inverted two-chamber bottle, containing a microbubble-forming preparation, with an aqueous solution in the lower chamber and solid and gaseous ingredients in the upper chamber.

-1313

Figure 4 illustrates the bottle of Figure 3, where the aqueous solution has been mixed with the solid ingredients to form microbubbles for administration to a patient.

Detailed description of the invention

As used herein, microbubbles are considered to be gas bubbles in an aqueous medium that have a diameter of between about 0.5 and 300 pm, preferably have a diameter of not more than about 200, 100, or 50 pm. Microbubbles may or may not have a layer or coating on a gas / liquid interface. If present, the coating may be one or more molecules thick. Additionally, the microbubbles can be trapped by a bimolecular layer (as in the case of laminar uni15 liposomes) or they can be trapped by several bilayer-type layers (multilamellar vesicles). These microbubbles may also be surrounded by more permanent shell-like structures, such as denatured proteins. Since emulsions are generally defined as a dispersion of two or more non-miscible fluids, stabilized by a surfactant interface, the gas dispersions of the present invention are essentially gas emulsions, with the discontinuous phase of the emulsion being a gas, rather than a liquid. Consequently, the term gas emulsion,

-1414 as used herein means a dispersion of a plurality of gas microbubbles in an aqueous medium.

For intravascular use, the optimal bubble size is determined by two competing interests. Minor bubbles are effective in circulating through small blood vessels and capillaries, but the echogenicity of ultrasound is strongly dependent on the size of the bubbles. Microbubbles suitable for enhancing vascular ultrasound contrast are therefore preferably about 1-10 pm in diameter, with 35 pm being preferred.

The present invention provides a gas emulsion or dispersion, in which the bubbles have a long longevity in vivo, and which is suitable for use as contrast enhancing agents by ultrasound or magnetic resonance imaging (MRI). Typical ultrasound contrast-enhancing agents exhibit a potential for contrast enhancement for only about one pass through the arterial system, or a few seconds to about a minute, and thus do not survive by passing the aorta in a patient, in followed by intravenous injection. In comparison, contrast agents, prepared in accordance with the present invention continue, to show lifetimes of increases in contrasts measured by

-1515 multiple passes through the entire circulatory system of a patient, followed by intravenous injection. The lives of bubbles of several minutes is easily demonstrated. Such elongation of the potential for contrast enhancement during ultrasound is highly advantageous. Furthermore, the contrast enhancing agents according to the invention provide superior imaging; for example, clear, vivid and distinctive images of the blood flowing through the heart, liver and kidneys are achieved. Thus, non-toxic doses can be administered into a peripheral vein and used to enhance images of the entire body.

While bubbles have been shown to be the most efficient ultrasound dispersants for use in intravenous ultrasound contrast agents, their main practical drawback is the extremely short lifetime of small suspended bubbles (typically less than 5 microns in diameter) , required to pass through the capillaries. This short life time is caused by increased gas pressure within the bubble, which results from surface tension forces on the bubble. This high internal pressure increases as the bubble diameter decreases. This increased internal gas pressure forces the gas into the bubbles to

-1616 dissolve them, resulting in crushing of the bubbles as the gas is forced into solution. Laplace's equation, ΔΡ = 2y / r (where ΔΡ is the increased gas pressure within the bubble, γ is the surface tension 5 of the bubble film a and r is the radius of the bubble a), describes the pressure exerted on a gas bubble from the surface or film surrounding the bubble. Laplace pressure is inversely proportional to the radius of the bubble; Thus, as the bubble shrinks, the pressure of Laplace 10 increases, increasing the rate of gas diffusion out of the bubble and the rate of shrinkage of the bubble.

In one embodiment, the present invention considers phospholipid surfactant-containing gas dispersion compositions that have certain advantages over other surfactants and other phospholipid-containing compositions. In a preferred embodiment, the composition includes two or more surfactants, which are selected to assist in the creation of a larger number of microbubbles and also to optimally reduce the surface tension at the gas / liquid interface) of the bubbles with the liquid. Furthermore, gases of low water solubility may advantageously constitute a part of the gas in the microbubbles. It has been found especially advantageous to use, in conjunction with a second higher surfactant

-1717 soluble in water, as a stabilizing combination of surfactants to improve gas trapping.

The stability of a gas emulsion is highly dependent on the properties to decrease the surface tension of the surfactant used as the emulsifying agent. Phospholipids, as they are known to function as the main component of the lung surfactant, are extremely efficient in this regard. They also easily form lamellar structures, such as bilayer sheets and liposomes, although this feature is not necessary to stabilize the gas dispersions of the present invention. Another determinant of the stability of the gas emulsion is the gas itself and the ability to stabilize by means of an osmotic effect, as described below. This combination results in a surprisingly stable and virtually useful microbubble.

Gas dispersion compositions according to the present invention can be prepared by spray drying an aqueous dispersion of a first surfactant, comprising a phospholipid, preferably at least one additional surfactant co-agent (also referred to herein as the second surfactant) and a hydrophilic monomer or polymer, or combinations thereof. The aqueous starting material may optionally include salts

-1818 and / or an inflation agent. Spray drying of such a solution, in accordance with the present invention, results in the production of a dry, hollow, approximately particulate, microspheric material.

Surprisingly, it has been found that a preformed spherical cavity including water soluble components (eg hydroxyethyl starch and its salts) and a relatively water soluble surfactant (eg Pluronic F-68, Tween 20 , dioctonylphosphotidyl-choline) and a phospholipid (eg, egg yolk phospholipid) when in the physical form produced by spray drying, they can form remarkably stable microbubbles when rehydrated. The surfactant need not be present in the liposomal or other lamellar form. This may be the result of the water first contacting the inner surface of the spherical cavity (0.5-10 microns in diameter), after filtering through the dissolving surfactants and structural agents, resulting in formation of a bubble of the desired size (the size of the cavity) initially surrounded by a saturated surfactant solution, and therefore having a maximally optimally packed surfactant coating. These bubbles are remarkably stable in vivo, even when

-1919 fill with water soluble gases (eg air or nitrogen).

This process, like the dry and reconstituted products thus obtained, are explained and described in more detail below.

Preparation of a Precursor Containing

Phospholipid

Dispersion:

For subsequent spray drying, a first aqueous solution was prepared, containing a hydrophobic phospholipid as a first surfactant and at least one additional, more hydrophilic surfactant. Preferably, the hydrophobic phospholipid has at least one acyl chain with a total of at least 10 carbon atoms (eg, a decanoyl phospholipid). In some embodiments, the first phospholipid surfactant has acyl chains of from about 10 or 14 to about 20 or 24 carbon atoms. For example, dipalmitoylphosphatidylcholine (comprising two acyl chains, each with 16 carbon atoms) can be used. The chain of. acyl can be hydrogenated or fluorinated. Other phospholipid headgroups are also considered. For example, phosphatidylserines, phosphatidylglycerols, or phosphatidylethanolamines will have properties suitable for the present invention.

Combinations of these phospholipids

-2020 may also comprise the first surfactant, such as naturally derived phospholipid products such as lecithin from egg or soy, or surfactants from the lung. Furthermore, the first phospholipid surfactant can be supplemented with other highly water-insoluble surfactants, such as sucrose ditri- and tetra-esters. Cholesterol can also supplement the first surfactant and has been found useful in promoting stability when supplied in a range of about 0.01 to 0.5 w / w cholesterol to phospholipid. Preferably, the acyl chains of the phospholipid are saturated, although unsaturated acyl groups are also within the scope of the present invention. The first surfactant is preferably provided in the range of about 0.005 to 20% w / v of the solution, more preferably in the range of 0.02 to 10% w / v.

The primary role of the first hydrophobic surfactant is to reduce the surface tension of microbubbles formed below equilibrium values. When relatively insoluble osmotic stabilizing gases are trapped (described in detail below), a first surfactant with very little water solubility is required, because the reduction in surface tension

-2121 below the equilibrium values is only possible when the surfactant diffuses more slowly than the trapped stabilizing gas. To achieve suitably low solubility of the surfactant, phospholipids with long acyl chains (i.e., comprising more than 10 carbon atoms) are particularly preferred.

The second surfactant is preferably more hydrophilic and diffuses faster than the long chain phospholipid comprising the first surfactant. The role of this second surfactant is that the formation of a stable gas dispersion is likely related to a faster dissolution rate in water reconstitution and greater effective gas entrapment, thus facilitating the creation of bubbles in the early stages of reconstitution, as further described below. In this way, the faster rate of diffusion of the second surfactant helps to create a relatively durable and continuous film, surrounding the gas in the reconstitution.

In the present invention, the preferred second surfactants can be selected from the group consisting of phospholipids, phosphocolines, lysophospholipids, nonionic surfactants, neutral or anionic surfactants, fluorinated surfactants, which can be neutral or anionic, and combinations of such

-2222 emulsifying or foaming agents. Some specific examples of surfactants, which are useful as the second surfactants, include polyoxypropylene and polyoxyethylene block copolymers (an example of such a class of compounds is Pluronic, such as Pluronic F-68), sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl-poly (ethylenoxy) ethanol, nonylphenoxypoly (ethylenoxy) ethanol, derived starches, hydroxy-ethyl-starch fatty acid esters, fatty acid salts, commercial food vegetable starches, dextran fatty acid esters, sorbitol fatty acid esters, gelatin, serum albumin and combinations thereof.

Also considered as the second surface active agent are polyoxyethylene fatty acid esters, such as polyoxyethylene stearates, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearate, polyethylene glycol ricinoleate glycol, ethoxylated soybean sterols, ethoxylated castor oils and their hydrogenated derivatives. Furthermore, nonionic alkyl glucosides, such as Tweens®, Spans® and Brijs® are also within the scope of the present invention. The Spans

-2323 include sorbitan, sorbitan tetraoleate, sorbitan triestearate tetraestearate, sorbitan tripalmitate, sorbitan trioleate, and sorbitan distearate. Tweens include polyoxyethylene sorbitan triestearate, polyoxyethylene tripalmitate, polyoxyethylene sorbitan trioleate. The family of

Brij is another useful category of materials, including polyoxyethylene stearyl ether

10. Anionic surfactants, particularly fatty acids (or their salts) having from 24 to 24 carbon atoms, can also be used. An example of a suitable anionic surfactant is oleic acid, or its salt, sodium oleate. Cationic surfactants and dodecyltrimethylammonium are their salts, such as chloride, also considered for use as second surfactants.

It will be appreciated from the above, that a wide range of second surfactants can be used. In reality, virtually any surfactant (including those to be developed) of higher solubility and water diffusivity than that of a longer chain phospholipid, comprising the first surfactant, can be used in the present invention. . The optimal surfactant for a given application can be determined through empirical studies that do not

-2424 require undue experimentation. Consequently, a practice in the technique of the present invention should select the surfactant based on such properties, such as biocompatibility. Use of a shorter chain phospholipid, which is more hydrophilic than the first phospholipid, has been found to be advantageous as a co-surfactant. As a specific example, a first phospholipid having acyl chains with 12 or 14 carbon atoms, can be provided with a second phospholipid as a co-surfactant, having acyl chains with eight to ten carbon atoms.

It has been found especially advantageous to provide a phospholipid comprising 12-carbon acyl chains as the first or second surfactants. For example, a phospholipid with 12-carbon acyl chains may comprise the first surfactant, and a sugar ester or pluronic compound may comprise the second surfactant. As another option, a 16-carbon acyl chain phospholipid may comprise the first surfactant, and a 12-carbon acyl chain phospholipid may comprise the second surfactant.

-2525

Furthermore, in order to have excellent bubble formation and persistence quality, the microbubbles formed with phospholipids, for both the former and the co-surfactant, have superior properties in the area of metabolic elimination after injection in vivo, as well as minimizing unwanted responses in vivo, such as complement activation, which can be a problem with prior art microbubbles.

Containing phospholipid surfactants, with acyl chains of 12 or 14 carbon atoms, appear to be especially advantageous in appearance. Microbubbles containing the phospholipid are believed to be not only more biocompatible than those containing surfactants without phospholipids, but also more biocompatible than liposomes. That is, they apparently evade the most effectively than the

<td>liposomes, and</td><td>So</td><td>are not</td><td>cleaned of</td><td colspan="2">circulation so</td>

<td>quickly.</td><td></td><td></td><td></td><td></td><td></td>

<td>How</td><td>I know</td><td>he pointed</td><td>with regard</td><td>to the first</td><td>agent</td>

<td>surfactant,</td><td>the</td><td>second</td><td>or co-agent</td><td>surfactant</td><td>can</td>

<td colspan="3">understand combinations</td><td>of the agents</td><td>surfactants</td><td>before</td>

described. Preferably, prior to spray drying, the second surfactant is supplied in the interval

-2626 from 0.005% to 20% by weight / volume. The first surfactant is not required to predominate the mixture. Either the first or the second surfactants can be provided in higher molarity and / or weight. In general, the total surfactant in solution is approximately 0.01% to 20% w / v of the solution.

Following production of the aqueous surfactant solution, as described above, an inflation agent, preferably a fluorocarbon, such as Freon 113, is added, creating a thick suspension. This inflation agent can be any material that turns into gas during the spray drying process. This inflation agent is then dispersed through the surfactant solution, for example a commercially available microfluidizer, at a pressure of 350 to 1050 kg / cm2. In a preferred embodiment of the present invention, a high-pressure homogenizer is used to obtain a conventional Freon 113 emulsion in a phospholipid-containing surfactant solution. This process forms a conventional emulsion comprising submicron Freon droplets not miscible with water, coated with a monomolecular layer of the surfactant. Dispersion with this and other techniques is common and well known to those skilled in the art.

-2727

The inclusion of an inflation agent in the solution to be spray dried results in a higher ultrasound signal per gram of spray dried powder, forming a larger number of hollow microspheres. The inflation agent nucleates the bubble formulation in the stream within the atomized droplets of solution entering the spray drier as these droplets mix with the hot air stream within the drier. Suitable inflation agents are those that supersaturate the solution within the gas or steam atomized droplets at the elevated temperature of the drying droplets (about 100 ° C). Suitable agents include:

one. Low boiling point solvents (below 100 ° C) dissolved, with limited miscibility with aqueous solutions, such as methylene chloride, acetone, and carbon disulfide, used to saturate the solution at room temperature.

2. A gas, for example CO2 or N2, used to saturate the solution at room temperature and high pressure (for example at 3 bar). The droplets are then supersaturated with the gas at 1 atmosphere and 100 ° C.

3. Non-miscible low-boiling (below 100 ° C) liquid emulsions, such as Freon 113, perfluoropentane, perfluorohexane, perfluorobutane,

-2828 pentane, butane FC-11, FC-11B1, FC-11B2, FC-12B2, FC21, FC-21B1, FC-21B2, FC-31B1, FC-113A, FC-122, FC-123, FC-132 , FC-133, FC-141, FC-141B, FC-142, FC-151, FC152, FC-1112, FC-1121 and FC-1131.

Inflation agents are added to the surfactant solution in amounts of approximately 0.5% to 10% by volume / volume of the surfactant solution. Approximately 3 volume / volume% of the inflation agent has been found to produce a spray-dried powder that forms suitable microbubbles. This inflation agent is substantially evaporated during the spray drying process and thus is not present in the final spray dried powder in more than trace amounts.

The aqueous precursor solution preferably includes a hydrophilic monomer or polymer or combinations thereof. This can be combined with the surfactant solution or, more preferably, form as a separate solution and combined with the surfactant precursor solution just prior to spray drying. The hydrophilic part can, for example, be a carbohydrate, such as glucose lactose or starch. Polymers, such as PVA or PVP are also considered for use with the present invention. Various starches and derived starches have been found especially suitable. Starches particularly

-2929 preferred for use in microbubble formation include those with a molecular weight greater than about 500,000 daltons or a dextrose equivalent value (DE) less than about 12. The value of the DE is a quantitative measure of the degree of hydrolysis of the starch polymer. It is a measure of reducing energy compared to a dextrose standard of 100. The higher the DE, the greater the extent of starch hydrolysis. Such preferred starches include food grade vegetable starches, of the type commercially available in the food industry, including those sold under the trademarks N-LOK and CAPSULE by National Starch and Chemical Co., (Bridgewater, NJ); derived starches, such as hydroxyethyl starch (available under the trademarks HETASTARCH and HESPAN from Du Pont Pharmaceuticals, Mhydroxyethyl starch from Ajinomoto, Tokyo, Japan). (Note that spray dried short chain starches can be used to produce microbubbles, but are not preferred, because they have a molecular weight of less than about 500.00 and do not stabilize the microbubbles. However, they can be used in the present invention in applications where no further stabilization is required.) The hydrophilic monomer or polymer is present in this embodiment of the precursor solution in the range of about 0.1 to 10%

-3030 w / v solution, with approximately 1 to

5% w / v having been found particularly suitable.

Other optional components of this embodiment of the precursor solution are various salts or other agents within the aqueous phase. These agents can include viscosity regulators, such as phosphate regulators or other conventional biocompatible regulators, or pH adjusting agents, such as acids or bases, osmotic agents (to deliver isotonicity, hypermolarity, or hypomolarity). Preferred solutions have a pH of about 7 and are isotonic. These additional ingredients each typically comprise less than 5% w / v of the solution. Examples of suitable salts include sodium phosphate (both monobasic and dibasic), sodium chloride, calcium phosphate, and other physiologically acceptable salts.

II. Spray Dried

The emulsion of the surfactant / inflation agent is preferably combined with a solution of the hydrophilic monomer / polymer and above, and dried by structures approximately its salts, of the type described sprayed to form a dry, hollow microsphere powder.

-3131

Commercially available spray dryers are well known to those of skill in the art, and the proper fit for any particular precursor solution can be readily determined through standard empirical testing, with reference to the examples that follow.

The Niro Portable Spray Dryer, used in Examples I-VII and IX-XII below, works by spraying a solution containing a surfactant with a two-fluid compressed air nozzle, which uses a high-speed jet of compressed air to break up the aqueous solution of the surfactant into droplets ranging from 2 to 20 microns in diameter. These droplets are then injected into a stream of hot air (typically 200 to 375 ° C) at the top of the drying chamber. The droplets in the surfactant solution are heated, almost instantly, to their boiling point of approximately 100 ° C. Although the evaporative coating prevents them from rising to a higher temperature, the temperature is even higher than the glass transition temperature of phospholipids and above the melting point of many other surfactants, such as Poloxamer 188 and Stearate of saccharose.

The water on the surface of the drop evaporates very quickly, causing the formation of compounds that

-3232 dissolve in the atomized solution. When the solution contains a hydrophilic polymer, such as hydroxyethyl starch (HES), a gel layer forms on the surface. Beneath the gel layer, a vapor bubble forms which inflates the gel sphere. As briefly described above, the presence of an inflating agent comprising a sparingly soluble, volatile solvent, such as methylene chloride or a volatile non-miscible solvent, such as Freon 113, provides nucleation sites for prompt bubble formation vapor, leading to increased steam inflation and thinner walled hollow spheres.

During the drying process, water either migrates through the pores in the gel layer to the surface of the sphere as a liquid, where it vaporizes or escapes from the sphere as vapor, through the same pores in the layer of gel. Finally, the water trapped in the HGES gel evaporates. During this phase of the drying process, evaporation and evaporative cooling are slower, and the sphere rises in temperature to correspond to the exit temperature of the spray drying chamber, typically at 100 to 120 ° C. The gel sphere shrinks as it dehydrates to supply hollow porous spheres of approximately 1 to 10 pm in diameter, with cover thicknesses of approximately 0.2 pm. The current

-3333 spray dryer exhaust air carries the spheres to a cyclone separator, where the powder is separated from the air stream by centrifugal force and is directed into the product container.

For various reasons, the composite structure of the dry spherical composition of the surfactant / polymer is characterized as random with the substantial absence of lamellar forms. First, homogenization to form an emulsion with an inflation agent, before spray drying deposits the surfactant in a monomolecular layer. Because drying takes place in a small fraction of a second, rapid entrapment of the surfactant in a concentrated polymer gel (for example HES), maintains the surfactants in approximately the physical state that they were prior to drying. Also, during the spray drying process, less water-soluble surfactants (sucrose stearate, long-chain phospholipids, etc.) are encouraged above their melting point or glass transition temperature, in the presence of a surfactant more soluble in water, and thus incorporated into the matrix of the hydrophilic polymer. This leads to more random surfactant structures.

This spray-dried composition, comprising gas-filled hollow microspheres, is an important product

-3434 of the present invention. This product provides significant advantages over the precursors that contain the lyophilized liposome. The spherical structure of the present microbubble precursor material is believed to serve to quickly and uniformly form the relatively incompressible, relatively insoluble, non-Newtonian surfactant film characterized by the preferred microbubbles of the present invention.

After spray drying is complete, the microspheres are packed in a container with an appropriate gas. This gas fills the microspheres and becomes the gas trapped in the microbubbles after reconstitution.

The various individual components of the microspheres preferably comprise the following proportions of the spray-dried final product, in% by weight:

<td>First phospholipid surfactant</td><td>0.05% to 90%</td>

<td>Second surfactant</td><td>0.05% to 90%</td>

<td>Hydrophilic structural material</td><td>1% to 99%</td>

<td>Sales, regulator, etc.</td><td>0% to 90%</td>

In particularly preferred embodiments, the composition has the following proportions, in% by weight:

-3535

<td>First phospholipid surfactant</td><td>0.1% to 10%</td>

<td>Second surfactant</td><td>0.1% to 10%</td>

<td>Hydrophilic structural material</td><td>10% to 60%</td>

Sales, regulator, etc.

10% to 60%

More preferably, the amount of the first surfactant (advantageously a phospholipid) is at least 1%, preferably at least 3%, 4%, or 5%, and more preferably at least 7%, 8%, or 10% of the total surfactant, by weight / weight. It can also constitute 25%, 50%,

75% or 95% of the total surfactant, weight / weight, and modalities lacking the second surfactant, while not preferred, are also considered.

In an alternative embodiment of the present invention, the composition of the precursor solution is such that the spray-dried liposome-forming powder is prepared. Such precursor solutions may have, for example, the composition of US Patent No. 5,380,519 to Schneider et al. , which is incorporated herein by reference. We have discovered that the spray drying technique, described here, to form the microbubble precursors of phospholipid-containing solutions is superior to lyophilization by Schneider et al., And that the resulting microbubbles appear to be much more stable than

-3636 those formed from the lyophilized material. Thus, in this aspect of the invention, it is considered that the spray drying technique can be used to prepare precursors that form the dry liposome, which is then reconstituted to form the microbubbles, as in Schneider et al ,, for use. in ultrasound imaging techniques.

III. Reconstitution and Selection of Gas

In reconstitution in an aqueous medium, the hydrophilic monomer or polymer which, in some preferred embodiments, supplies the structure to the shell, like salts and any regulators, etc., that may be present, dissolves rapidly and separates leaving behind is a gas emulsion or dispersion comprising gas bubbles that are surrounded by a layer of the surfactant which is left behind. The first phospholipid surfactant and the most hydrophilic surfactant co-agent, are hypothesized to perform different functions.

Without limitation to any particular theory of operation, the second surfactant (or co-surfactant) is apparently useful in aiding the dissolution of water-soluble structural materials, and may also diffuse rapidly enough in reconstitution to heal the surfactant from free voids, which exist during the dissolution of the cover. It has been

-3737 found that reconstitution of the microspheric powder containing, in part a relatively water-soluble surfactant, in conjunction with a more hydrophobic surfactant, produces a much higher number of bubbles per 5 millimeter of intensifying agent than reconstitutes the powder than contains only a single hydrophobic surfactant, although the second relatively hydrophobic surfactant, Apparently important in the transition from the dried hollow sphere to the gas bubble coated with the surfactant, the most hydrophobic first phospholipid is thought to be the most effective stabilizing agent after the bubbles are formed.

The gas dispersion thus created is therefore fundamentally different from the previous contrast-enhancing compositions 15 containing the phospholipid. As described above, Ryan and Unger gas-containing liposomes do not involve surfactant layers at the bubble gas / liquid interface, but essentially involve pure bubbles trapped in the aqueous nuclei of the liposomes. This can be distinguished from the gas emulsion or microbubble dispersion of the present invention, in that it appears (without being limited to any particular theory of operation) that small gas bubbles are surrounded by a relatively non-evanescent layer

-3838 durable, surfactant with orientation such that the hydrophilic head groups associate with the aqueous liquid, and the hydrophobic tail groups associate with the dispersed gas bubbles.

Likewise, in contrast to Schneider et al. , the gas dispersions of the present invention do not require the presence in solution of the liposomes or other lamellar structures of surfactants. Indeed, gas dispersions with excellent in vivo stability can be prepared in accordance with the present invention, in which the surfactants used are unable to form liposomes. The absence of lamellar structures of surfactants in solution does not mean that they affect the efficacy of the contrast agents of the present invention. Additionally, it has been observed that the presence of a fluorocarbon osmotic stabilizer gas in the bubbles dramatically increases the stability of the gas dispersions of the present invention, while the presence of a fluorocarbon osmotic stabilizer has little effect on the stability of the microbubbles produced from lyophilized liposomes. These differences in behavior further suggest ((again, without being limited to any particular theory of operation) that the gas in the bubbles of the present invention is trapped by a relatively durable, non-evanescent layer of

-3939 surfactant with orientation such that the hydrophilic head groups are associated with the aqueous liquid, and the hydrophobic tail groups are associated with the dispersed gas bubbles.

Suitable bubbles containing air, nitrogen, or other gases normally present in the blood can be created by reconstituting the spray-dried microspheres, described above, in an aqueous medium. It has also been found that the life of the bubbles can be improved when a relatively water-insoluble gas, such as a fluorocarbon, is made to permeate the dried microspheres prior to reconstitution. In this case, the invention will use a first gas or gases (a primary modifying gas) which, optionally, is ordinarily present in normal blood and serum, in combination with one or more additional second gases (a gas osmotic agent or agents or a secondary gas) that act to regulate the osmotic pressure within the bubble. Through regulation of the osmotic pressure of the bubble, the gas osmotic agent (defined herein as a single entity or mixture of chemical entities) exerts pressure within the bubble, helping to prevent deflation. Optionally, the modifier gas can be a gas that is not ordinarily present in blood or serum. However, the modifier gas must be capable of diluting and

-4040 maintaining the gas osmotic agent or agents at a partial pressure below the vapor pressure of the gas osmotic agent or agents, while gases in the blood or other surrounding liquid diffuses into the bubble. In an aqueous medium, water vapor is not considered to be one of the '' gaces cuestiónη question. Similarly, when the microbubbles are in a non-aqueous liquid medium, the vapor from such a medium is not considered to be one of the gases.

We have discovered that by adding a gas osmotic agent having, for example, reduced membrane permeability through the surface or reduced solubility in the external continuous liquid phase, the life of a bubble formed with it can be increased.

This result is achieved through the entrapment, within the chosen gas emulsion, of a combination of gases, preferably a primary modifying gas or gas mixture that will dilute a gas osmotic agent at a partial pressure less than the vapor pressure of the agent. gas osmotic, until the modifier gas is exchanged with the gases normally present in the external medium. The gas osmotic agent or agents are generally relatively hydrophobic and relatively impervious to the bubble membrane and also possess the ability to develop gas osmotic pressures greater than 50, 75, or 100 Torr. In

-4141 In a preferred embodiment, the gas vapor pressure of the gas osmotic agent is preferably less than 760 Torr at 37 ° C, preferably less than about 750, 740, 730, 720, 710 or 700 Torr, and in some modalities, less than 650, 600, 500 or 400 Torr. In preferred embodiments, the vapor pressure of the primary modifying gas is at least 660 Torr at 37 ° C and the vapor pressure of the gas osmotic agent is at least 100 Torr at 37 ° C.

The first gas and the second gas are respectively present in a molar ratio of about 1: 100, 1:75, 1:50, 1:30, 1:20, or 1:10, up to about 1000: 1, 500: 1, 250: 1, 100: 1, 75: 1 or 50: 1, and where the first gas has a vapor pressure of at least about (760 x) mm Hg at 37 ° C, where x is the vapor pressure of the second gas at 37 ° C and where the vapor pressure of each of the first and second gases is greater than about 75 or 100 mm Hg at 37 ° C.

Gas emulsion or gas dispersion bubbles, prepared in accordance with a preferred embodiment of the invention, may also possess appropriate additional advantages. In one embodiment, mixtures of non-osmotic gases with osmotic stabilizing gases (or gas osmotic agents) are used to stabilize the resulting bubble size distribution during and immediately after

-4242 production. In bubble generation, the higher Laplace pressure in the smaller bubbles causes diffusion through the liquid phase to the larger Laplace lower pressure bubbles. This causes the mean size distribution to increase above the 5 micron capillary dimension limit over time. This is called disproportion. When a mixture of a non-osmotic gas (for example air) is used with an osmotic vapor (for example CgF ^), a slight reduction in volume of minor bubbles, due to the air leaving the bubble, concentrates the osmotic gas and increases its osmotic pressure thus slowing further shrinkage, while large bubbles increase slightly in volume, diluting the osmotic gas and retarding further growth.

An additional advantage of using a mixture of extremely blood-soluble gases (for example, 87.5% by volume of CO2) and an osmotic gas mixture (for example, 28% CgF vapor] ^ + 72% air) is the one that, when injected, these bubbles shrink rapidly due to the loss of CO2 θη the blood. The bubbles, in the injection, will experience a decrease of 87.5% in volume due to the loss of CO2. This loss of CO2 corresponds to half the diameter of the bubble. Therefore, one can prepare large diameter bubbles (for example from 9 pm)

-4343 using simplified mechanical means that will shrink below 5 microns in injection. In general, such a gas emulsion was initially prepared when the first gas is present in a ratio of at least 1: 1 to the second gas, preferably at least 3: 2, 2: 1, 3: 1, 4: 1 , 5: 1 or 10: 1,

When the microbubble membrane is more permeable to the first gas than to the second gas (for example, the membrane has respective gas permeabilities in a ratio of at least 2: 1, 3: 1, 4: 1, 5: 1 or 10: 1, preferably even larger, for example 10: 1, 40: 1 or 10 :: 1), the bubbles advantageously shrink from their first original diameter to a second average diameter of 75% or less of their original diameter quite rapidly (for example within one, two, four, or five minutes). Then, when at least one relatively membrane-permeable gas is present in the aqueous medium comprising the continuous phase of the gas emulsion, the bubble is preferably stabilized at or around the second diameter for at least about 1 minute, preferably 2, 3, 4 or 5 minutes. In a preferred embodiment, the bubbles maintain a size between about 5 or 6 pm and 1 pm for at least 1, 2, 3, 4, or 5 minutes, stabilized by a differential osmotic pressure of the gas. The gas tension in the external liquid is preferably at least about 700 mm Hg. Also a gas

-4444 relatively impermeable to the membrane is also in the microbubble to create such an osmotic pressure differential.

As noted above, the gas osmotic agent is preferably a gas which is less permeable through the bubble surface than the modifier. It is also preferable that the gas osmotic agent be less soluble in blood and serum. Therefore, it will now be understood that the gas osmotic agent can be a gas at room or body temperature or it can ordinarily be a liquid at body temperature, as long as it has sufficient partial or vapor pressure at the use temperature. to deliver the desired osmotic effect.

Therefore, fluorocarbons or other non-gas compounds at room or body temperature can be used, provided they have sufficient vapor pressure, preferably at least about 50 or 100 Torr at body temperature or , more preferably, at least about 150 or 200 Torr. It will be noted that when the gas osmotic agent is a mixture of gases, the relevant measure of vapor pressure is the vapor pressure of the mixture, not necessarily the vapor pressure of the individual components of the mixed gas osmotic agent.

-4545

It is also important when using a perfluorocarbon as the osmotic agent within a bubble, the particular perfluorocarbon does not condense at the partial pressure present in the bubble and at body temperature.

Depending on the relative concentrations of the modifying gas causing it to be secondary.

bubble pressure

Primary primary laplace and agent can shrink and concentrate gas osmotic, gas quickly bubble gas osmotic agent

Such an osmotic shrinkage of gas equals the occurrence of the maximum absolute blood pressure (pressure) of the bubble minus the tension of plus to external than that of air, or the saturation tension of air, of blood (essentially an atmosphere). Thus the condensing partial pressure of the resulting gas mixture at 37 ° C must be above the equilibrium partial pressure, discussed above, of the osmotic agent.

A list of some compounds that possess adequate solubility and vapor pressure criteria is provided in Table I:

TABLE I Perfluoro Propanes, ΟβΕθ Perfluoro Butanes, C4F1Q Perfluoro Cyclobutanes, C4F3

-4646 Perfluoro Pentanes, C5F12 Perfluoro Cyclopentanes, C5H1Q Perfluoro Methylcyclobutanes, C5F1Q Perfluoro Hexanes, CgF ^ Perfluoro Cyclohexanes, CgF ^ 2 Methyl Cyclopentanes, Perfluoro, CgF ^ 2 Dimethyl-Cyclobutanes, PerfluoroFluorines C7F16 perfluoro cycloheptanes, C7H14 perfluoro methyl cyclohexanes, C7H14 perfluoro dimethylcyclopentanes, C7F14 perfluoro trimethylcyclobutanes, C7F14 perfluoro triethylamines, N (02 ^) 3

As will be appreciated, one of ordinary skill in the art can readily determine other compounds that perform adequately in the present invention that do not meet the solubility and pressure criteria described above. Rather, it will be understood that certain compounds may be considered outside the preferred range in their solubility or vapor pressure, if such compounds compensate for such aberration in the other category and provide superior insolubility or high vapor pressure.

-4747

It should also be noted that for medical uses, gases, both the modifier gas and the gas osmotic agent, must be biocompatible or not physiologically detrimental. Eventually, the microbubbles containing the gas phase will decompose and the gas phase will be released into the blood, either as a dissolved gas or as submicron droplets of condensed liquid. It will be understood that gases will be removed primarily from the body through lung respiration or through a combination of respiration and other pathways in the reticuloendothelial system.

A surprising discovery was that mixtures of PFCs, for example C4F10 (as a combination of the modifier gas and a gas osmotic agent) saturated with CgF ^ vapor (as the main gas osmotic agent), can stabilize bubbles by times longer than any single component. This is because C4F10 is a gas at body temperature (and thus can act both as a modifier gas and as a gas osmotic agent) it has somewhat reduced membrane permeability and is only slightly soluble in the C5F14 at body temperature. In this situation, the osmotic gas pressures of the two agents add to each other, leading to increased persistence of bubbles above that of the

-4848 air / CgF ^ only in mixture. It is only possible that the dew point of the longer molecular weight CgF] ^ component, which persists longer, is increased, allowing a higher maximum osmotic pressure of the gas to be exerted. Other PFC blends will perform similarly. Preferred mixtures of the PFCs will have ratios of 1:10 to 10: 1 and include such mixtures as perfluorobutane / perfluorohexane and perfluorobutane / perfluoropentane. These preferred fluorine chemicals can be branched or straight chain.

As discussed before, we have also discovered that mixtures of non-osmotic gases in combination with the gas osmotic agent, act to stabilize the size distribution of the bubbles, before and after injection. In bubble generation, the higher Laplace pressures in the smaller bubbles cause diffusion through the liquid phase to the larger bubbles at the lower Laplace pressure. This causes the average size distribution to increase above the 5 micron capillary dimension limit over time. This is called a disproportion.

However, when a mixture of a modifier gas (for example, air or carbon dioxide) is used with an osmotic gas agent (for example C ^ F] ^), a slight

-4949 reduction in volume of the smallest bubbles, due to one of the modifying gases left by the bubble, will concentrate the osmotic gas and increase its osmotic pressure, thus delaying further shrinkage. On the other hand, the larger bubbles will increase slightly in volume, diluting the osmotic gas and also retarding further growth.

Therefore, we have discovered that through the use of a gas that is relatively hydrophobic and has relatively low membrane permeability, the rate of bubble decomposition can be reduced. Thus, by reducing the rate of bubble decomposition, the half-lives of the microbubbles are improved and the potential for contrast enhancement is extended.

The desired gas is made to permeate the dried microspheres, placing these microspheres inside a bottle, which is introduced into a vacuum chamber to evacuate the air. The air is then replaced with the desired gas or combination of gases (a preferred combination of gases is perfluorohexane-saturated nitrogen at 13 ° C). The gas will then diffuse into the holes in the spheres. Diffusion can be aided by pressure or vacuum cycles. The bottle is then corrugated and preferably sterilized with gamma radiation.

-5050

It will be appreciated that kits can be prepared for use in obtaining microbubble preparations of the present invention. These kits may include a container that locks up the gas or gases, described above (to form the microbubbles), the liquid, and the surfactant. The container can contain all sterile dry components, and the gas, in a chamber, with the liquid sterile aqueous in a second chamber of the same container. Suitable two-chamber flask-type containers are available, for example, under the trademarks WHEATON RS177FLW or S-1702FL, from Wheaton Glass Co., (Millville; NJ). Such containers are illustrated in Figures 1-4. Referring to Figures 1 and 2, the illustrated Wheaton container 5 has an upper chamber 20, which can contain an aqueous solution 25, and a lower chamber 30, which can contain the dry ingredients 35 and a desired gas. A shutter 10 is provided, which separates the upper chamber from the environment, and a seal 15 separates the upper chamber 20 from the lower 30, which contains the spray dried, hollow microspheres 35 (powder), and the gas osmotic agent. . Pressing the shutter 10 pressurizes the relatively incompressible liquid, which pushes the seal 15 down into the lower chamber 30. This releases the aqueous solution 25 into the lower chamber 30, causing it to

-5151 which results in dissolution of the powder 35 to form the stabilized microbubbles 45, which contain the trapped gas osmotic agent. Excess gas osmotic agent 40 is released from lower chamber 30 into upper chamber 20. This arrangement is user-friendly and has the unexpected added benefit of sealing the small amount of the water-impermeable gas osmotic agent in the lower chamber by covering the inter-chamber seal with a thick (1.27 to 3.175 cm) layer of the aqueous solution and the advantage that this aqueous solution can be introduced into the lower chamber without raising the pressure in the powder chamber by more than 10%. Thus, there is no need for pressure ventilation. (In contrast, conventional reconstitution of a solute in a single-chamber vial with a needle and syringe without ventilation can result in the production of considerable intra-chamber pressure, which could collapse the microbubbles.

Alternatively, an inverted two-chamber bottle can be used for the preparation of microbubbles. Referring to Figures 3 and 4, the same bottle is used, as described above, except that the plug 50 is elongated, so that it dislodges the inner seal 15 when it is depressed. In this microbubble preparation method, the hollow microspheres 35, dried by

-5252 sprayed, and gas osmotic agent 40, are contained within upper chamber 20. Aqueous solution 25 and gas osmotic agent 40 are contained within lower chamber.

30. When the shutter 50 is depressed, it dislodges the seal 15, allowing the spray dried hollow microspheres to mix with the aqueous solution 25 in the presence of the gas osmotic agent 40. An advantage associated with this method of microbubble formation is that the aqueous phase can be instilled first and sterilized by autoclaving or other means, followed by instillation of the spray dried microspheres. This will prevent potential microbial growth in the aqueous phase prior to sterilization.

Although a particular dual chamber container has been illustrated, other suitable devices are known and commercially available. For example, a two-chamber glass syringe, such as the Dual Chamber prefilled syringe system, BD HYPAK Liquid / Dry 5 + 5 ml (Becton Dickinson, Franklin Lakes, NJ; described in US Patent No. 4,613,326) can be advantageously used to reconstitute spray dried powder. The advantages of this system include:

one. Convenience of use;

-5353

2. The gas osmotic agent, insoluble in aqueous media, is sealed by a chamber of the aqueous solution on one side and an extremely small area of the elastomer seals the needle on the other side; and

3. A filter needle, such as Monoject # 305 (Sherwood Medical, St. Louis MO), can be fitted into the syringe at the same time as it is manufactured, to ensure that no undissolved solids are injected. The use of the two-chamber syringe to form the microbubbles is described in Example XIV.

One of ordinary skill in the art will appreciate that other two-chamber reconstitution systems, capable of combining spray-dried powder with the aqueous solution in a sterile manner, are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the water-insoluble osmotic gas and the environment, to increase the shelf life of the product. When the material necessary to form the microbubbles is no longer present in the container, it can be packaged with the other components of the kit, preferably in the form of a container adapted to facilitate prompt combination with the other kit components.

-5454

Examples of particular uses of the microbubbles of the present invention include perfusion imaging of the heart, myocardial tissue, and determination of perfusion characteristics of the heart and its tissues during stress or exercise tests, or defects or perfusion changes due to myocardial infection. Similarly, myocardial tissue can be seen after oral or venous administration of drugs designed to increase blood flow to tissue. Also, the visualization of changes in myocardial tissue due to or during various interventions, such as vein grafting of coronary tissue, coronary angioplasty, or the use of thrombolytic agents (TPA or streptokinase), can also be increased. As these contrast agents can be conveniently administered via a peripheral vein to enhance visualization of the total circulatory system, they will also aid in the diagnosis of general vascular pathologies and in the ability to monitor the viability of placental tissue ultrasonically.

However, it should be noted that these principles have application beyond ultrasound imaging. Actually, the present invention is broad enough to encompass the use of emulsions

-5555 gas containing phospholipids in any system, including non-biological applications.

It will also be understood that other components can be included in the microbubble formulations of the present invention. For example, osmotic agents, stabilizers, chelating agents, regulators, viscosity modulators, air solubility modifiers, salts and sugars, can be added to modify microbubble suspensions for maximum life and efficacy of contrast enhancement. Such considerations as sterility, isotonicity, and biocompatibility can govern the use of such conventional additives to injectable compositions. The use of these agents, as will be understood by those of ordinary skill in the art, and the specific amounts, ratios, and types of agents can be determined empirically, without undue experimentation.

Various embodiments of the present invention provide surprising advantages. Spray-dried starch formulations provide prolonged stability in the bottle, particularly when the molecular weight of the starch is greater than 500,000. Fatty acid esters of sugars, such as sucrose monostearate, as well as block copolymers, such as Pluronic F-68 (with a hydrophilic / lipophilic balance (HLB)

-5656 greater than 12) allow the powder to bubble the instant it rehydrates. Spray-dried formulations with a structural agent, such as starch, starch derivatives, or dextrin, deliver a significantly lower total dose of the surfactant than comparable sonicated formulations. The use of two-chamber vials of water, which provide an additional gas osmotic agent seal, provide increased shelf life, and increased convenience of use. Spray-dried formulations with a structural agent (such as starch or dextrin), a hydrophobic phospholipid, and a more water-soluble surfactant co-agent provide gas emulsions with greatly increased in-vivo half-lives.

Any of the microbubble preparations of the present invention can be administered to a vertebrate animal, such as a bird or a mammal, as a contrast agent for ultrasonically imaging portions of this vertebrate animal. Preferably the vertebrate is a human being and the portion from which the image is formed is the vasculature of the vertebrate. In this embodiment, a small amount of microbubbles (eg, 0.1 ml / kg to 12 mg / kg of the spray dried powder), based on the body weight of the vertebrate) is introduced intravascularly into the animal. Other amounts of microbubbles, such

-5757 as about 0.005ml / kg to 1.0ml / kg, can be used. Imaging of the heart, arteries, veins, and blood-rich organs, such as the liver and kidneys, can be done ultrasonically with this technique.

The following description will be more fully understood with reference to the following Examples. However, these examples are illustrative of the preferred methods of practicing the present invention and do not limit the scope of the invention or the appended claims.

Example I

Spray Drying of a Solution Containing Phospholipids

One liter of the following solution in water was prepared for injection: 2.0% w / v Maltointrin Maltrin M-100, (Grain Processing Corp. Muscatine, IA), 0.95% w / v Sodium Chloride (Mallinckrodt, St. Louis; MO), 1.0% of Superonic F-68 (Serva, Heidelberg, Germany), 1.0% in w / v of Ryoto Sucroise Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo Japan) and 0.5% of hydrogenated phospholipid, Lipoid E-100-3, (Lipoid, Ludwigshafen, Germany).

This solution was then spray dried in the Niro Atomizer Portable Spray Dryer, equipped with a two-fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which uses the following settings:

-5858 hot air flow rate inlet air temperature outlet air temperature

Atomizer Air Flow Liquid Feed Rate

1,119 m3/ min

245 ° C

100 ° C

350 liters / min liter / hour

The dry, hollow spherical product had a diameter between about 1 µΜ and 15 µΜ and was collected in a cyclone separator, as is normal for this dryer. Aliquots of powder (250 mg) were weighed into 10 ml tubular flasks, evacuated and sprayed with perfluorohexane-saturated nitrogen, at 13 ° C, and sealed. Nitrogen was saturated with perfluorohexane by passing it through three gas wash bottles filled with perfluorohexane, immersed in a 13 ° C water bath.

In the reconstitution with 5 ml of water for injection, numerous bubbles were observed under a light microscope, ranging in size from 1 to 20 microns. The fact that many bubbles of about 1 micron could be observed for an appreciable time, demonstrates that the added stability gained by the inclusion of a phospholipid in the formula, as an additional non-Newtonian viscoelastic surfactant.

-5959

Example II

Comparison of phospholipid emulsions vs. sucrose ester gas emulsions

One liter of each of the following four solutions was prepared with water for injection:

Solution 1:

3.9% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.25% w / v sodium chloride (Mallinckrodt. St. Louis, MO)

2.83% sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.42% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

Solution 2:

2.11% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

0.32% w / v of Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei

Food Corp., Tokyo, Japan)

0.16% w / v of Ryoto Sucrose Stearate S-570 (Mitsubishi-Kasei

Food Corp., Tokyo, Japan)

Solution 3

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6 % sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

-6060

Solution 4:

0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

0.45% w / v Phosphothidylcholine from Egg, Hydrogenated, EPC-3 (Lipoid, Ludwigshafen, Germany)

Solutions 2 and 4 were added to a high cut mixer and chilled in an ice bath. A 3.0% v / v coarse suspension of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) was obtained in the liter of 10 solutions 2 and 4. These suspensions were emulsified using a microfluidizer ( Microfluidics Corporation, Newton, MA, model M-110F) at a pressure of 700 kg / cm2, a temperature of 5 ° C, for 5 passes. The resulting emulsion 4 was added to solution 3 and the resulting emulsion 2 was added to solution 1. The mixture of Formulas 1 and 2 (containing the sucrose ester surfactant) and the mixture of Formulas 3 and 4 (containing the phospholipid surfactant) were then spray dried in a Niro Atomizer Portable Spray drying apparatus. Dryer, equipped with a 20 two-fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which uses the following settings:

Mix of Formulas 1 and 2:

hot air flow rate 878 liters / min inlet air temperature 370 ° C

-6161 exhaust air temperature

Atomizer Air Flow Liquid Feed Rate

Mix of Formulas 3 and 4 hot air flow rate inlet air temperature outlet air temperature

Atomizer Air Flow Liquid Feed Rate

120 ° C

290 liters / min

1.5 liters / hour

878 liters / min

325 ° C

120 ° C

290 liters / min

1.5 liters / hour

The dry, hollow, spherical product had a diameter between approximately 1 µΜ and 15 µΜ and was collected in a cyclone separator as is common for this dryer. Aliquots of powder (250 mg) were weighed into 10 ml tube vials, sprayed with perfluorohexane saturated nitrogen at 13 ° C and sealed. The nitrogen was saturated with perfluorohexane by passing it through three perfluorohexane filled gas wash bottles, immersed in a 13 ° C water bath.

The vials were reconstituted with 5 ml of water for injection, after inserting an 18 gauge needle as a vent to relieve pressure as the water was injected. One ml of the resulting microbubble suspension was injected intravenously into a rabbit of

-6262 approximately 3 kg of weight, instrumented to monitor the Doppler ultrasound signal of your carotid artery. A 10 MHz flow cuff (Triton Technology, Inc., San Diego, CA; model ES-10-20) connected to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF Doppler signal to a Lecroy 9410 oscilloscope (LeCroy, Chesnut Ridge, NY). The mean square root (RMS) voltage of the signal computed by the oscilloscope was transferred to a computer and the resulting curve adjusted to obtain the peak echogenic signal intensity and the half-life of the microbubbles in the blood. Signals before contrast were less than 0.1 volt RMS.

While the sucrose ester formulation produced an initial ultrasound scatter signal 29% greater than the signal from the phospholipid formulation due to the increased concentration of microbubbles, surprisingly, the persistence of the phospholipid formulation was

<td colspan="5">substantially higher. The sign of</td><td>the formula of</td><td>ester</td><td>of</td>

<td>saccharose</td><td>decreased</td><td>to the</td><td>30 of</td><td>its</td><td>original sign</td><td>in</td><td> 140</td>

<td>seconds,</td><td>While</td><td>the</td><td>formula</td><td>of</td><td colspan="2">phospholipids lasted</td><td> 550</td>

seconds before decreasing to the 30% signal level, demonstrating superior persistence of a formula that uses a phospholipid as the non-Newtonian viscoelastic surfactant.

-6363

Example III

Comparison of microbubbles in the formulation of the water-insoluble phospholipid vs. microbubbles the formulation of the phospholipid / water soluble surfactant, insoluble in water (Poloxamer 188)

One liter of each of the following emulsions was prepared by spray drying, as described in Example II:

Formulation A: Phospholipid Formulation, Insoluble in Water

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45% w / v of egg phospholipids, hydrogenated E PC 3 (Lipoid, Ludwigshafen, Germany)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ).

Formulation B: Phospholipid / Water Soluble Formulation, Insoluble in Water (Poloxamer 188)

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

-6464

0.45% w / v of egg phospholipids, hydrogenated E PC 3 (Lipoid, Ludwigshafen,

Germany)

0.45% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

In reconstitution of 100 mg of spray-dried powder of Formulation A with 5 ml of water, approximately 20 million bubbles per ml were observed, ranging in size from 1 to 20 pm. In the reconstitution of 100 mg of spray-dried powder of Formulation B with 5 ml of water, approximately 315 million bubbles (1575% more bubbles than Formulation A9 per ml were observed, ranging in size from 1 to 20 p.m.

The addition of a relatively water-soluble surfactant [HLB (Poloxamer 188) = 29-0] to a water-insoluble surfactant in the microbubble formulations, significantly increased the concentration of the bubbles formed, leading to a contrast agent of more effective ultrasound. The (HLB) is a number between 0 and 40 assigned to emulsified agents and substances that emulsify. HLB is indicative of emulsification behavior and is related to the balance between the hydrophilic and lipophilic portions of the molecule (Rosen, M., (1989), Surfactants and Interfacial Phenomena, Second Edition, John Wiley & Sons, New York, pages 326-329).

-6565

Example IV

Comparison of microbubbles of the formulation of the water-insoluble phospholipid vs. the microbubbles of the phospholpxdo / water-soluble, water-insoluble formulation (Polysorbate 20)

One liter of each of the following emulsions was prepared for spray drying, as described in Example II:

Formulation A: Phospholipid formulation insoluble in water

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, basic (Mallinckrodt. St Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45% w / v of hydrogenated egg phospholipids E PC 3 (Lipoid, Ludwigshafen,

Germany)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

Formulation B: Phospholipid / water soluble formulation, insoluble in water (Polysorbate 20)

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

-6666

0.45% w / v of hydrogenated egg phospholipids E PC 3 (Lipoid, Ludwigshafen,

Germany)

0.15% w / v of Polysorbate 20 (ICI, Wilmington, DE)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

In the reconstitution of 100 mg of the spray-dried powder of Formulation A with 5 ml of water, approximately 20 million bubbles per ml were observed, ranging in size from 1 to 20 pm. In the reconstitution of 100 mg of the spray-dried powder of Formulation B with 5 ml of water, approximately 250 million bubbles per ml were observed (1150% more bubbles than in Formulation A), ranging in size from 1 at 20 pm.

In conclusion, the addition of the relatively water-soluble surfactant, polysorbate 20 [HLB = 16.7] to the water-insoluble surfactant, the hydrogenated phosphatidylcholine in the microbubble formulations, significantly increased the concentration of the bubbles formed, leading to a more effective ultrasound contrast.

Example V

Prepared gas emulsion, with the combination of phospholipids

One liter of the following emulsion was prepared for spray drying, as described in Example II:

-6767

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.22% w / v of dipalmitoylphosphatidylcholine (Genzyme, Cambridge, MA)

0.31% w / v of dioctanoylphosphatidylcholine (Avanti Polar Lipids, Albaster, AL) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

In the reconstitution with 5 ml of water, approximately 51 million bubbles per ml were observed, ranging in size from 1 to 20 microns. The constant decrease in echogenic signal for this microbubble formulation was determined to be 0.0029 (1 / sec).

One milliliter of this formulation was injected into the ear vein of a 2.5 kg New Zealand White Rabbit. An image of the rabbit was subsequently taken with an Acuson 128xP-5 ultrasound scanner, equipped with a 5MHz transducer. In the infusion, the echogenicity of the blood vessels and chambers of the heart was intense and persisted for several minutes. Furthermore, the echogenicity of the myocardium and a solid organ, such as the liver and kidney, was homogeneously intense and persisted for several minutes. Notably, the echogenicity of the portal and hepatic veins were isointense, indicating minimal uptake by reticuloendothelial phagocytic cells from the

-6868 liver, resulting in prolonged vascular persistence.

Example VI

Biocompatibility of gas emulsions prepared from long chain / short chain mixed phospholipids

One liter of the following emulsion was prepared for spray drying, as described in Example II.

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.22% w / v dipalmitoylphosphatidylcholine (Syngene Ltd., Cambridge, MA)

0.31% w / v dioctanoylphosphatidylcholine (Avanti Polar Lipids Inc., Albaster, AL)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Scíence, Gibbstown, NJ)

In these ratios of dipalmitoylphosphatidylcholine to dioctanoylphosphatidi-choline, the surfactants formed mixed micelles only. In the reconstitution with 5 ml of water, approximately 51 million droplets of gas emulsion per ml were observed, ranging in size from 1 to 20 microns. The first-order decrease constant of the echogenic signal of the gas emulsion in rabbits at a dose of 5 mg / kg was determined to be

-6969

0.0029 sec ”l. This corresponds to an intravascular half-life of 4 minutes.

The gas emulsion was tested for complement activation using an in vitro C3a diagnostic kit, supplied by Quidel Corp. (San Diego, CA). No difference was observed between the gas emulsion and the negative (saline) control. F indicating that the gas emulsion does not activate the complement. It is well known that the complement activates the microbubbles with the naked eye.

Sample Tested [C3a] (ng / ml)

Zymosan (positive control) 43403

Saline solution (negative control) 604

Gas emulsion412

The gas emulsion was also tested for changes in hemodynamics in anesthetized dogs at a dose of 20 mg / kg. No change in mean arterial pressure or pulmonary artery pressure was observed. These results indicate that there are no hemodynamic effects with the gas emulsion at 10,100 times the clinically relevant dose.

-7070

<td>Time (minutes)</td><td>Average Blood Pressure (mm Hg)</td><td>Pressure Pulmonary artery (mm Hg)</td>

<td colspan="2">Thus, a</td><td>excellent efficacy and</td>

biocompatibility in the same gas emulsion formulation.

Example VII

Gas emulsion containing phospholipids supplemented with cholesterol

Half a liter of each of the following solutions was prepared in water for injection. Solution 1 contains starch and salts and Solution 2 contains phospholipids and cholesterol dissolved in a mixture of Freon 113 and ethanol. Solution 2 was added to a high cut mixer and chilled in an ice bath. A thick suspension of 1,1,2-trichlorotrifluoroethane (Freon 113) was obtained by adding half a liter of water with vigorous stirring. This

-7171 suspension was emulsified with Solution 2, as described in Example II. The resulting emulsion was added to Solution 1 to produce the following spray drying formula:

3.6% w / v m-HES, hydroxyethyl (starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Malllnckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.22% w / v dipalmitoylphosphatidi-choline (Genzyme., Cambridge, MA)

0.31% w / v dioctanoylphosphatidylcholine (Avanti Polar Lipids Inc., Albaster, AL)

0.05% w / v Cholesterol (Sigma, St. Louis, MO)

2.4% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

0.6% v / v ethanol (Spectrum Chemical, Gardena, CA).

This emulsion was then spray dried in a Niro Atomizer Portable Spray apparatus.

Dryer, equipped with a two-fluid atomizer (Niro

Atomizer, Copenhagen,

Denmark), which uses the following approximate adjustments:

hot air flow rate inlet air temperature outlet air temperature atomizer air flow

878 liters / min

325 ° C

120 ° C

200 liters / minute feed rate of the emulsion 1.5 liters / hour.

-7272

The dry, hollow, spherical product had a diameter between approximately 1 µΜ and 15 µΜ and was collected in the cyclone separator as is normal for this dryer. Aliquots (100 mg) were weighed into 10 ml tube flasks, sprayed with perfluorohexane saturated nitrogen at 13 ° C and sealed as in Example II. The vials were reconstituted with 5 ml of water for injection, after inserting an 18 gauge needle as ventilation to relieve pressure as the water was injected. A 0.25 ml / kg dose of the resulting microbubble suspension was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal of its carotid artery, again, as in Example

II. The signal, 1 minute after injection, was 0.71 volt with a decrease constant of 0.010 sec ~ ^. Hematology samples were taken during the first 60 minutes after injection. There was no detectable drop in platelet count or detectable complement activation according to Example VI.

Example VIII

Efficacy in vivo of reconstituted lyophilized liposomes

A liposome-forming solution, with a total lipid concentration of 50 mg / ml, was prepared with hydrogenated soy lecithin (S PC-3, Lipoid, Ludwigshafen,

-7373

Germany), and dicetyl phosphate (Sigma, St. Louis, MO) in a 9: 1 molar ratio. Following the Szoka and Papahadjopoulos Reverse Phase Evaporation Method in Proc. Nat. Acad., Sci. 75 USA (1978), 4194, the surfactants were dissolved in 120 ml of a 1/1 v / v solution of diethyl ether / chloroform. 40 ml of deionized water were added. The mixture was sonicated for 10 minutes at 0-4 ° C with a 3mm probe sonicator (50W, Vibra Cell, Sonics & Meteriais Inc., Danbury CT) to form an emulsion. A liposome dispersion was formed by removing the solvent under reduced pressure by rotary evaporation and filtration of the solution through a 1.0 pm polycarbonate filter at 65 ° C. Fractions of 1 ml of the liposome solution were then mixed with 4 ml of a 15% w / v maltose solution (Sigma, St. Louis) in 10 ml ultrasound bottles, frozen at -30 ° C and lyophilized (FTS Systems, Stone Ridge, NY). The flasks were gasified with nitrogen or perfluorohexane-saturated nitrogen at 13 ° C. The lyophilized powder was reconstituted in 5 ml of water, at the following concentrations:

12.0% w / v Maltose (Sigma, St. Louis, MO)

0.926% w / v of S PC-3 Hydrogenated Soy Lecithin (Lipoid, Ludwigshafen, Germany)

0.072% w / v Dicethyl Phosphate (Sigma, St. Louis, MO)

-7474

One ml of the resulting microbubble suspension was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal from its carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA; model ES-10-20) connected to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF Doppler signal to an oscilloscope LeCroy 9410 (LeCroy, Chesnut Ridge, NY). The mean square root (RMS) voltage of the signal computed by the oscilloscope was transferred to a computer and the resulting curve adjusted to obtain the peak intensity of the echogenic signal and the half-life of the microbubbles in the blood. Signals before contrast were less than 0.1 volt RMS.

Neither nitrogen nor perfluorohexane gasified liposome-containing formulations showed significant or lasting echogenicity in the rabbit model.

Example IX

Effect of perfluorohexane gasification on the ultrasound efficacy of lyophilized liposome formulations vs. spray-dried gas emulsion formulations

-7575

One liter of each of the following emulsions was prepared for spray drying, as described in Example II:

Formulation A: Microbubble formulation of

Sucrose Ester

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45% w / v sucrose ester 11025003 (Alliance Pharmaceutical Corp.,

San Diego, CA)

1.95% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

Formulation B: Phospholipid Microbubble Formulation

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt St. Louis, MO)

2.6% w / v sodium phosphate, basic di (Mallinckrodt. St Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45% w / v dipalmitoyl phosphatidylcholine (Genzyme, Corp. Cambridge, MA)

0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

-7676

Formulation C: Lyophilized Liposome Formulation

Approximately 40 ml of a dispersion of liposomes containing hydrogenated soy lecithin and dicetyl phosphate (9: 1 molar ratio) at a total lipid concentration of 50 mg / ml in water, were prepared using the Phase Evaporation Method Reverse (REV), described by Szoka and Papahadjopoulos (see Example VIII)

The formulation is summarized as follows:

<td></td><td>Formula of</td><td>Reconstituted</td>

<td></td><td>Dry powder</td><td>for injection</td>

<td>Hydrogenated Soy Lecithin (Lipoid S PC-3, Lipoid,</td><td> 7.14</td><td> 0.918</td>

<td>Ludwigshafen, Germany)</td><td></td><td></td>

<td>Dicethyl Phosphate (Sigma, St. Louis, MO)</td><td> 0.55</td><td> 0.072</td>

<td>Maltose (Sigma, St Louis, MO)</td><td> 92.3</td><td> 12.0</td>

<td>Water for injection</td><td></td><td>5.0 mi</td>

<td>Two jars were prepared</td><td>decade</td><td>one of the</td>

<td>formulations described above;</td><td>one is</td><td>gasified with</td>

one perfluorohexane-nitrogen mixture, the other contained only nitrogen. Samples (6 total) were reconstituted with 5 ml of water and evaluated for efficacy using a Rabbit Model Doppler Signal Enhancement apparatus.

Pulsed. The doses administered to the rabbit were 5 mg of

-7777 dry powder per kg of the rabbit, for formulations A, B and C, respectively.

The echogenic signals at 60 sec for the formulations that do not contain perfluorohexane, A, B and C, were 0.040, 0.142 and 0.005 V, respectively, the echogenic signals at 60 sec. for their respective formulations containing perfluorohexane they were 1,232, 0.826 and 0 V. In conclusion, the addition of a perfluorohexane gasification step did not significantly increase the efficacy of ultrasound (defined here as the 60-second echogenic signal) of the lyophilized liposome formulation. Both the efficacy of the spray dried sucrose ester microbubble and perfluorohexane containing phospholipid microbubble formulations were increased by 2980 and 482%, respectively. Thus, fundamental differences in structure and behavior exist between the gas emulsions of the present invention and the microbubble preparations obtained from lyophilized liposomes.

Example X

Efficacy of the spray-dried dispersion containing the water-insoluble phospholipid described in Example 4 of US Patent No. 5,380,519 to Schneider, et al.

A formulation containing the proportions of dicetyl phospholipid and phosphate, as described in

-7878

Example 4 of US Patent No. 5,380,519 to Schneider et al., Was prepared by spray drying the following emulsion. Surfactants were not laminated (converted to liposomes) as in the Schneider example.

One liter of each of the following solutions was prepared with water for injection: Solution 1, containing the starch and salts and Solution 2 containing the surfactants. Solution 2 was added to a high cut mixer and chilled in an ice bath. A thick suspension of 1,1,2-trichlorotrifluoroethane (Freon 113) was obtained in 1 liter of Solution 2. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-110F) at 700 kg / cm2, 5 ° C for 5 passes. The resulting emulsion was added to Solution 1 to produce the following spray drying formula:

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.058% w / v Dicetyl Phosphate (Sigma, St. Louis, MO)

0.742% w / v of Phospholipid PC-3 (Lipoid, Ludwigshafen, Germany)

3.0% v / v of 1,1,2-tnclorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

-7979

This emulsion was then spray dried in a Niro Atomizer Portable Spray Dryer apparatus, equipped with a two-fluid atomizer (Niro Atomizer, Copenhagen, Denmark), using the following approximate settings: rate of hot air flow = 878 liters / min inlet air temperature = 325 ° C outlet air temperature = 120 ° C atomizer air flow ~ 290 liters / min emulsion feed rate = 1.5 liters / hour

The dry, hollow, spherical product had an approximate diameter of 1 µΜ to 15 µΜ and was collected in the cyclone separator as is normal for this dryer, aliquots of powder (100 mg) were weighed into 10 ml tube vials , sprayed with nitrogen alone or with nitrogen plus perfluorohexane (PFH) and sealed as in the previous examples.

-8080 Doppler ultrasound signal from your carotid artery, as in the previous examples.

Signals were observed with and without PFH. The agent containing the PFH produced a signal of 0.08 volt in 60 seconds, with a signal of 0.01 volt in 200 seconds. The nitrogen-only agent produced a 0.2 volt signal in 60 seconds with a 0.4 volt signal in 200 seconds. Since this formula does not contain a more water-soluble surfactant, the signals are much lower than in the previous examples. The spray drying process, however, presents this mixture of non-laminar surfactants in a physical state, which produced detectable signals, unlike the laminar formula of Schneider et al. in Example 4, as demonstrated in Example VIII above. This formula also differs from the other examples in this application in that the addition of perfluorohexane reduced the resulting signal rather than greatly increased it.

Example XI

Microbubbles that do not contain fluorocarbon

Two formulations (A, mixed phospholipids and B phospholipid + Poloxamer 188) were prepared by spray drying the following emulsions with a similar process.

-8181

One liter of each of the following solutions was prepared with water for injection: Solution 1 containing starch and salts and Solution 2 containing surfactants. Solution 2 was added to a high cut mixer and chilled in an ice bath. A coarse suspension of 1,1,2-trichlorotrifluoroethane (Freon 113) was obtained in 1 liter of Solution 2. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-110F) at 700 kg / cm2, 5 ° C for 5 passes. The resulting emulsion was added to Solution 1 to produce the following spray drying formula:

Formula A (Mixed Phospholipids)

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.22% w / v of dipalmitoylphosphotidallcholine (Genzyme., Cambridge, MA)

0.31% w / v dioctinoyl-phosphotidal-choline (Genzyme, Cambridge, MA)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

Formula B (Phospholipid + Poloxamer 188)

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

-8282

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.15 p / v of Poloxamer 188 (BASF, Parsipany, NJ)

0.45% w / v Phospholipid PC-3 (Lipoid, Ludwigshafen, Germany)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

This emulsion was then spray dried in a Niro Atomizing Portable Spray Dryer apparatus, equipped with a two-fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which employs the following approximate settings:

hot air flow rate - 878 liters / min inlet air temperature = 325 ° C outlet air temperature ~ 120 ° C atomizer air flow = 290 liters / min emulsion feed rate - 1.5 liters / hour

The dry, hollow, spherical product had a diameter of between about 1 and 15 µΜ and was collected in the cyclone separator as usual for this dryer. Aliquots of powder (100 mg) were weighed into 10 ml tube jars, sprayed with nitrogen only, and sealed.

The vials were reconstituted with 5 ml of water for injection, after inserting an 18 gauge needle as a vent to relieve pressure as the water was injected. A mi of the suspension resulting from

-8383 microbubbles were injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppier ultrasound signal from its carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA; model ES-10-20) connected to a System 6 Doppier flow module (Triton Technology Inc.) fed the RF Doppier signal to a LeCroy oscilloscope 9410 (LeCroy, Chesnut Ridge, NY). The mean square root (RMS) voltage of the signal computed by the oscilloscope was transferred to a computer and the resulting curve adjusted to obtain the intensity of the echogenic signal and the half-life of the microbubbles in the blood.

Significant signals were observed with both formulas. Formula A produced a signal of 0.25 volt in 60 seconds with a signal of 0.13 volt in 200 seconds. Formula B produced a 0.3 volt signal in 60 seconds with a 0.2 vol signal in 200 seconds. Phospholipid-free formulas produced only background signals when treated in the same way.

As described above, this may be the result of the water first making contact with the inner surface of the spherical cavity (0.5-10 microns in diameter), after filtering through the dissolving surfactants and structural agents that

-8484 result in the formation of a bubble of the desired size (the size of the cavity) that is initially surrounded by the saturated surfactant solution and therefore has an optimally packaged maximum surfactant coating, increasing the trapped gas These bubbles are remarkably stable in vivo even when filled with water soluble gases (eg air or nitrogen).

Example XII

Effect of phospholipid acyl chain length on echogenic efficacy of ultrasound

One liter of each of the following emulsions was prepared for spray drying, as described in Example II:

Formulation A: Dimiristoyl-Phosphatidylcholine formulation

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45 w / v dimyristoyl phosphatidylcholine (Genzyme Corp., Cambridge, MA)

0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

-8585

Formulation B: Distearoyl-Phosphatidylcholine formulation

3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan)

3.0% w / v Sodium Chloride (Mallinckrodt. St. Louis, MO)

2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO)

0.39% w / v Sodium Phosphate, Monobasic (Mallinckrodt. St. Louis, MO)

0.45% w / v distearoyl-phosphatidyl-choline (Genzyme Corp., Cambridge, MA)

0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ)

3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ)

After reconstitution with 5 ml of water, the two formulations were evaluated for efficacy, using a Rabbit Model apparatus to Increase the Pulsed Doppler Signal, as in Example II, and the echogenic signal measured as a function of time. The doses administered to

<td colspan="2">rabbit were 5 mg of the</td><td>powder</td><td>dry</td><td>by</td><td>kg of</td><td colspan="3">rabbit.</td>

<td>Time (seconds)</td><td> 20</td><td> 60</td><td> 100</td><td> 200</td><td> 300</td><td> 400</td><td> 500</td><td> 600</td>

<td>Formulation A - Signal Echogenic (V)</td><td> 0.8</td><td> 0.6</td><td> 0.5</td><td> 0.4</td><td> 0.4</td><td> 0.4</td><td> 0.3</td><td> 0.2</td>

<td>Formulation B - Signal</td><td> 0.5</td><td> 0.4</td><td> 0.2</td><td> 0.2</td><td> 0.1</td><td> 0.1</td><td> 0.1</td><td> 0.1</td>

Echogenic (V)

The echogenic signal, as a function of time, was on average higher for the formulation containing dimyristoyl-phosphatidylcholine (DMPC) than for the formulation containing distearoyl-phosphatidylcholine (DSPC). Both

-8686 fatty acid ester chains of DMPC contain 14 carbon atoms, while fatty acid ester chains are 18 carbon atoms in length for DSPC. This difference in chain length between the two phospholipid compounds results in a different gel at the crystal-liquid phase transition temperature. At temperatures above this transition temperature, the hydrocarbon chains are in the molten state and the phospholipids form a liquid crystal phase. This transition temperature is 55.5 ° C for DSPC and 23.5 ° C for DMPC in water.

Therefore, the use of a first phospholipid surfactant which is in the liquid crystal state after injection (rabbit body temperature = approximately 37.5 ° C) may be advantageous.

Example XIII

Microbubble Formation with the Use of a Two-Chamber Flask

<td></td><td>800 mg of</td><td>powder</td><td>spray dried were weighed</td><td>in</td>

<td colspan="2">the lower chamber</td><td>of a</td><td>two-chamber bottle of 20</td><td>me</td>

<td>Wheaton</td><td>RS-177FLW</td><td>(Figure</td><td>one). The jar was filled</td><td>with</td>

<td>nitrogen</td><td>saturated</td><td colspan="2">perfluorohexane at 13 ° C, before</td><td>of</td>

<td>insert</td><td>the seal of</td><td colspan="2">the internal chamber. The upper chamber</td><td>I know</td>

<td colspan="3">filled with 10 ml of water</td><td>sterile for injection.</td><td>The</td>

-8787 upper chamber shutter was inserted to eliminate all air bubbles in the upper chamber. By depressing the upper shutter, the seal between the chambers was forced into the lower chamber, allowing water to flow into the lower chamber and reconstituting the powder (Figure 2). Numerous stable microbubbles formed, as demonstrated by the light microscope. This procedure demonstrated the convenience of this form of packaging and the elimination of the need to provide ventilation to eliminate pressure build-up, when the aqueous phase is added to the powder.

Example XIV

Microbubble formation with the use of a double chamber syringe

One hundred mg of the spray dried powder was weighed into a 5 ml + 5 ml HYPAK Liquid / Dry dual chamber syringe (Becton Dickinson, Franklin Lakes, NJ) and shaken into the powder chamber (end of needle). The inter-chamber seal was then placed just above the diversion channel. A needle, containing a 5 µΜ filter, was then mounted on the syringe. The chamber containing powder was then filled with the gas osmotic agent, placing the assembly in a vacuum chamber, evacuating and filling the chamber with the gas osmotic agent, the nitrogen saturated with per

-8888 fluorohexane, at 13 ° C. The filter needle allowed the evacuation and filling of the atmosphere in the chamber containing the powder. A cover of the sealing needle was then placed over the needle. The liquid chamber was then filled with 4 ml of water for injection and the plunger was seated using temporary ventilation (a wire inserted between the glass syringe barrel and the plunger, to thereby remove all air bubbles.

• To reconstitute, the needle seal cover was removed to remove pressure build-up in the powder chamber. The plunger was then depressed, forcing the inner chamber seal to the offset position, allowing water to flow around the inner chamber seal into the chamber containing the powder. The movement of the plunger stopped when all the water was inside the powder chamber. The syringe was shaken to dissolve the powder. Excess gas and any large bubbles were expelled by holding the syringe with the needle end up and further depressing the plunger. The solution containing numerous stabilized microbubbles (as observed by the light microscope) was then ejected from the syringe, pressing the plunger to its limit.

The foregoing description details certain preferred embodiments of the present invention and describes the best mode

-8989 considered. However, it will be appreciated that regardless of how detailed the above text may appear, the invention can be practiced in many ways and must be interpreted in accordance with the appended claims, and any equivalents thereof.

-9090

Contents4

74 members in 19 offices

This recordMember, by publication datePriority claim9 more offices are in the list below

Priority claims1

Priority claims
Document	Office	Kind	Date
39568095	United States of America	A	1995-02-28

Members74

Family members, oldest first
Document	Office	Kind
CA2164813A1	Canada	A1	1995-02-09
WO9503835A1	World Intellectual Property Organization (WIPO)	A1	1995-02-09
AU7478294A	Australia	A	1995-02-28
EP0711179A1	European Patent Office (EPO)	A1	1996-05-15
CA2212113A1	Canada	A1	1996-09-06
WO9626746A1	World Intellectual Property Organization (WIPO)	A1	1996-09-06
AU4922196A	Australia	A	1996-09-18
JPH09501164A	Japan	A	1997-02-04
US5605673A	United States of America	A	1997-02-25
US5626833A	United States of America	A	1997-05-06
US5639443A	United States of America	A	1997-06-17
NO973489D0	Norway	D0	1997-07-30
FI973496A	Finland	A	1997-08-26
FI973496A7	Finland	A7	1997-08-26
FI973496L	Finland	L	1997-08-26
NO973489L	Norway	L	1997-08-27
US5695741A	United States of America	A	1997-12-09
EP0812214A1	European Patent Office (EPO)	A1	1997-12-17
PL321952A1	Poland	A1	1998-01-05
US5720938A	United States of America	A	1998-02-24
CN1182373A	China	A	1998-05-20
KR19980702548A	Republic of Korea	A	1998-07-15
AU694135B2	Australia	B2	1998-07-16
US5798091A	United States of America	A	1998-08-25
MX9706402AThis record	Mexico	A	1998-08-30
AU8933898A	Australia	A	1999-01-14
HU9802095A2	Hungary	A2	1999-01-28
HUP9802095A2	Hungary	A2	1999-01-28
JPH11501305A	Japan	A	1999-02-02
HU9802095A3	Hungary	A3	1999-03-29
HUP9802095A3	Hungary	A3	1999-03-29
HK1013403A1	Hong Kong, China	A1	1999-08-27
AU731099B2	Australia	B2	2001-03-22
AU731671B2	Australia	B2	2001-04-05
US6258339B1	United States of America	B1	2001-07-10
US6280704B1	United States of America	B1	2001-08-28
US6280705B1	United States of America	B1	2001-08-28
AU5199701A	Australia	A	2001-08-30
US6287539B1	United States of America	B1	2001-09-11
US2002028179A1	United States of America	A1	2002-03-07
US2002031476A1	United States of America	A1	2002-03-14
US6372195B1	United States of America	B1	2002-04-16
US2002051750A1	United States of America	A1	2002-05-02
US2002054854A1	United States of America	A1	2002-05-09
US2002098151A1	United States of America	A1	2002-07-25
AU767705B2	Australia	B2	2003-11-20
KR100407755B1	Republic of Korea	B1	2004-02-18
US2004033199A1	United States of America	A1	2004-02-19
US6706253B2	United States of America	B2	2004-03-16
JP3559849B2	Japan	B2	2004-09-02
EP0711179B1	European Patent Office (EPO)	B1	2004-11-03
AT281183T	Austria	T	2004-11-15
ATE281183T1	Austria	T1	2004-11-15
US2004228801A1	United States of America	A1	2004-11-18
DE69434119D1	Germany	D1	2004-12-09
DK0711179T3	Denmark	T3	2005-02-14
PT711179E	Portugal	E	2005-03-31
ES2231775T3	Spain	T3	2005-05-16
EP1550464A1	European Patent Office (EPO)	A1	2005-07-06
US6939531B2	United States of America	B2	2005-09-06
JP2005263804A	Japan	A	2005-09-29
US6953569B2	United States of America	B2	2005-10-11
DE69434119T2	Germany	T2	2005-11-03
US2005244338A1	United States of America	A1	2005-11-03
US2005281747A1	United States of America	A1	2005-12-22
US7005120B2	United States of America	B2	2006-02-28
US7141235B2	United States of America	B2	2006-11-28
CA2212113C	Canada	C	2007-04-24
JP4229918B2	Japan	B2	2009-02-25
CA2164813C	Canada	C	2009-11-24
EP0711179B2	European Patent Office (EPO)	B2	2010-09-01
CN1182373B	China	B	2010-12-08
ES2231775T5	Spain	T5	2011-02-02
DE69434119T3	Germany	T3	2011-05-05

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	Event	Code
2013-08-31	Abandonment or withdrawalAbandonedFA	FA
2006-03-31	Transfer or rightsGB	GB

Stabilized gas emulsion containing phospholipid for ultrasound contrast enhancement.

Abstract

Term

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

-1 STABILIZED GAS EMULSION. WHAT CONTAINS PHOSPHOLIPIDS

TO INCREASE THE CONTRAST OF THE ULTRASOUND

BACKGROUND OF THE INVENTION

Priority claims1

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Titles2

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