WO1993023013A1

WO1993023013A1 - Extravascular system for infusion of soluble substances

Info

Publication number: WO1993023013A1
Application number: PCT/US1993/004761
Authority: WO
Inventors: Ralph K. Ito; Frank W. Logerfo; Arthur J. Sytkowski
Original assignee: New England Deaconess Hospital
Priority date: 1992-05-20
Filing date: 1993-05-19
Publication date: 1993-11-25
Also published as: AU4382593A

Abstract

Specified substances are introduced into the bloodstream of an animal by placing them in a particular type of fluid reservoir residing in the body of the host animal and allowing the fluid from the reservoir to join the bloodstream via the lymphatic system or by transudation across a graft of material that has been inserted in a blood vessel. The fluid reservoir is formed from serous fluid that has transudated from the bloodstream across the grafted material to the extravascular space and pooled in a region formed by the body in response to the presence of the transudate. The specified substance can be incorporated in the fluid of the fluid reservoir by a variety of processes including secretion of the substance from recombinant cells adherent to a fibrous network inside the reservoir. An assemblage, that includes the blood vessel graft, the particular type of fluid reservoir and a source of the specified substance, provides the capability of introducing the specified substance into the bloodstream.

Description

EXT AVASCULAR SYSTEM FOR INFUSION OF SOLUBLE SUBSTANCES

Description

Background of the Invention

All animals, including man, have a continual need for substances from extracellular sources in order to remain viable. These substances are obtained from external sources or are biochemically synthesized by cellular metabolic processes. Many of these substances are secreted from the cells in which they are produced and delivered to their destination through extracellular fluid. If the transit distance is relatively long, in terms of cellular dimensions, the substances are usually transported via the blood- stream. The delivered substances can be any of a wide variety of substances, including inorganic ions, small organic molecules, metal-organic molecules or biological macro- molecules. They can be fats, polyols, enzymes, structural proteins, polypeptides, amino acids, complex saccharides, simple sugars, organic acids, organic bases, hormones or factors.

Normally, the animal incorporates these substances from the environment or synthesizes and secretes them in adequate quantities to sustain life free from disease. However, sometimes a need arises to artificially supple- ment the amount of these substances by supplying them or their precursors from an external, non-natural source. When the precursors are artificially administered, the expectation is that they will be metabolized to form the sought substances.

Needs also arise, to sustain relatively disease-free life, for the external supply of drugs to the animal. These drugs may be administered for a short period or for long durations of time. The purpose of these drugs is to alleviate a disease or the symptoms of such disorders or, alternatively, to prevent a disease from occurring.

Substances can also be administered from external sources to achieve desired effects unrelated to any disease. These effects are of potential benefit to the recipient or person administering the substance. For example, hormones can be given to domesticated animals to increase milk production or to rapidly increase body weight.

Until now, the artificially administered substances have been delivered to the individual animal from some type of defined container. If orally administered, the substance is embedded in a capsule, tablet or powder, or, alternatively, is a liquid from a suitable container. If administered through the airway of the individual animal, the substance originates from an inhaler or similar suitable container. If administered through the skin of the individual animal, the substance initially resides in a patch adhesively applied to the skin or in a hypodermic syringe or other suitable container that can be attached to a device adapted for delivery of the substance through an orifice in the body of the individual animal.

In some circumstances, a reservoir containing the substance may be surgically implanted within the body cavity of the individual animal. The reservoir is made of a synthetic material such as silicone or polytetra- fluoroethylene (PTFE) . The reservoir holds a pre¬ determined quantity of the substance and may release this substance by slow effusion or by a preprogrammed delivery schedule. The release rate is established prior to implantation and depends on the needs of the recipient or controlling person. The reservoir may be located at any designated site within the recipient individual. The criteria for placement of the reservoir normally are proximity to the target tissue for which the implanted substance is intended, surgical accessability, minimi¬ zation of invasive surgical procedures, and minimization of impairment of anatomical or physiological function of the recipient following the reservoir implantation.

Fulfillment of these criteria is not easily achieved. The fulfillment of one criterion often occurs only with the concomitant sacrifice of other criteria. The implantation of a reservoir near the desired target tissue usually requires surgical precision and displacement of organs and tissues from their normal locations. For example, the implantation of a reservoir containing insulin at the site of the pancreas requires a surgical incision through muscle and abdominal fascia and displacement of the small intestine. Conversely, the implantation of a reservoir at a peripheral location avoids the surgical trauma and minimizes tissue displacement but the release of the substance from the reservoir is remote from the target tissue and more substance must be released to provide the desired effect since the concentration of the substance is diluted as it travels through the extracellular fluid from the reservoir to the target tissue.

Delivery of the substance from the surgically im- planted reservoir is limited by the reservoir design and by the location of the reservoir in the recipient's body. Reservoirs can be fabricated to release the contained substance by passive diffusion from the external surface of the reservoir or from a defined orifice in the reservoir.

If a reservoir which releases the substance by passive diffusion from its external surface is used, the amount or directionality of its release cannot be con¬ trolled. The substance is not efficiently transmitted from the reservoir to the target tissue because it is released to the immediate environment surrounding the reservoir which is the extracellular space. The substance must then reach the target tissue by some undesignated process. If a reservoir which releases the substance from a defined orifice is used, the reservoir must be surgically positioned so its orifice is in proximity with the target tissue for maximum substance transmission efficiency. Although this reservoir positioning provides efficient transmission of the substance from the reservoir to the target tissue, the accompanying surgical procedure is nontrivial and the reservoir orifice may become dislodged from its intended position by the relative movement of the surrounding tissues during voluntary or involuntary movement of these tissues. An implanted reservoir can contain only a limited amount of the desired substance. This amount is dictated by the reservoir configuration and the substance con¬ stitution. The substance is continually subject to degradation in the reservoir as well as elsewhere in the recipient animal body other than at the target tissue. The intended metabolic and unintended target tissue degradation of the substance continues until the substance supply is depleted. Once depleted of the substance, the reservoir is useless unless it is replaced by another reservoir containing the substance, usually by a surgical procedure. Each implanted reservoir is, therefore, only functional for a limited time period.

The use of an implanted reservoir containing the desired substance also has another disadvantage. Since the reservoir is made of a synthetic material, there may be a rejection reaction to it by the recipient host animal. The reaction can be immunologic or allergic in nature or it can be encapsulated or, in some instances, excreted from the recipient animal. The usefulness of the implanted reservoir is thereby impaired. The rejection reaction additionally causes discomfort to the recipient animal and requires otherwise utilizable metabolic resources.

Summary of the Invention

This invention pertains to the introduction of a specified substance into the bloodstream of an animal, such as a human, by providing a source of the substance in an extravascular reservoir of transudate fluid which has formed outside an artificial material that has been grafted into a blood vessel of the animal. The transudate fluid is serous fluid from the bloodstream of the animal that has penetrated the grafted artificial material and formed the transudate fluid reservoir. The substance introduced into the transudate fluid reservoir is then transported to the bloodstream of the animal as the transudate fluid is transported to the bloodstream.

The invention also pertains to an assemblage for introducing a specified substance into the bloodstream of an animal such as a human. The assemblage is composed of an artificial material that has been grafted into an existing blood vessel of the animal, an extravascular reservoir of transudate fluid and a source of the specified substance. The grafted artificial material has the property of permitting serous fluid to penetrate it so transudation of the serous fluid can occur between the bloodstream in blood vessels and the extravascular space. Transudation can occur either from the interior of the blood vessel to the extravascular region or from the extravascular region to the interior of the blood vessel.

The invention uses the extravascular reservoir of transudate fluid to incorporate the specified substance for subsequent transport via the bloodstream to the target tissue. Since the bloodstream ultimately reaches almost all tissues, there is no need for a specific externally supplied device for transporting the specified substance from its source to the target tissue. Also, since the transudate fluid is derived from the blood sera of the animal, it is not immunologically rejected by the host ani al. The specified substance can be supplied by any source, including autologous biological cells. The biological cell source can survive and flourish in the transudate fluid, which is a biocompatible, nourishing medium, so an unlimited supply of the substance can exist and be utilizable. The extravascular reservoir need not have a defined shape or demarcated anatomical limit. Thus, its formation is as compatible with the relative positions of the host animal's tissues as possible. That is, the extravascular reservoir only minimally displaces these tissues. The extravascular reservoir is not restricted to a limited number of anatomical sites in the body of the host animal. Since the presence of the grafted artificial material initiates formation of an extravascular reservoir by the transudation of serous fluid from the bloodstream through the grafted material, the extravascular reservoir location is directly related to the location of the grafted material. Wherever such a graft can be placed, a contiguous extravascular reservoir can be formed.

Brief Description of the Figure

The Figure is a photograph of a Dacron graft showing an adherent external capsule and cut Dacron fibers enmeshed in fibrin.

Detailed Description of the Invention

in this invention, a specified substance is intro¬ duced into the bloodstream of an animal by initially placing or producing the substance in an extravascular fluid reservoir of transudate fluid located within the recipient animal's body. The transudate fluid that contains the specified substance then moves into the bloodstream. The animal can be any biological organism that has a bloodstream with anatomically defined blood vessels. This, of course, includes humans.

The specified substance can be of any type that is soluble in the fluid of the fluid reservoir. This in¬ cludes ions, inorganic compounds and organic compounds. Often, the specified substance will be a polypeptide or steroid hormone or a biological factor, a drug or a metabolic precursor to a metabolic process. The substance can be an amino acid, a polypeptide or protein, a steroid, a nucleic acid or derivative of a nucleic acid, a polyol, a fatty acid or a derivative of a fatty acid, a fat, an amine or a polyamine, a vitamin, a sugar or carbohydrate, or a phosphorylated compound.

The fluid in the fluid reservoir is serous in nature. That is, this fluid is blood sera that has left the bloodstream and pooled in an anatomical region of the host animal's body. Blood sera is the fluid portion of blood from which the particulate material, such as blood cells and platelets, has been removed. This fluid contains all the substances normally found in the blood other than particulate material. These substances include nutrients as well as ions that maintain physiologically compatible extracellular pH and osmolarity conditions suitable for viability of cells. These attributes of the fluid are important for incorporation and transport of the specified substance because the specified substance, when presented to target tissues, is presented to these tissues in a natural environment. These pH and osmolarity attributes are particularly important when the specified substance is produced within the fluid reservoir by cells, which remain viable in the environment provided by the fluid and can continue to produce the specified substance for their natural lifetime.

The fluid of the fluid reservoir originates from the bloodstream of the host animal. This fluid reaches the fluid reservoir by penetrating an artificial material that has been grafted into an existing blood vessel of the host animal. Blood sera leaves the bloodstream, passes through the grafted artificial material to the extravascular region of the blood vessel contiguous to where the artificial material has been grafted and then continues to the fluid reservoir. The process of fluid movement through or across the grafted artificial material from one side to the other is called transudation. Usually, the direction of transudation is from the lumen of the blood vessel to the extravascular region. However, transudation can occur in the opposite direction, i.e., from the extravascular region through grafted artificial material to the lumen of the blood vessel. When transudation does occur in this direction, the transudate often includes the specified substance.

The fluid that emerges from the grafted artificial material after transudation is called the transudate. Usually, transudate refers to the fluid that has passed from the lumen of the blood vessel through the grafted artificial material to the extravascular region outside the grafted artificial material. However, the term can refer to the fluid that moves across the grafted arti¬ ficial material in the opposite direction, then often including the specified substance. Thus, the transudate may or may not include the specified substance. The fluid that has just passed from the lumen of the blood vessel through the grafted artificial material to the extra¬ vascular region normally does not include the specified substance. The transudate fluid that flows from the fluid reservoir through the grafted artificial material to the lumen of the blood vessel, though, often includes the specified substance. In both situations, the fluid is referred to as the transudate since it has undergone the process of transudation. Usually, the transudate is substantially serous in constitution.

The artificial material that has been grafted into an existing blood vessel of the host animal through which transudation occurs can be made of any material that allows the process of transudation. The term "artificial" is used in the sense that the artificial material is not naturally formed or located at the site to which it has been grafted. This material normally is compatible with the host animal's immune system so rejection of the grafted material does not occur. The material is composed of substances that can provide functional grafts in blood vessels of the host animal. These grafts allow blood to continue to flow through the grafted blood vessel relatively unimpeded and are strong enough to withstand the pressure differentials and dynamics between the inside and outside of blood vessels. The material should be at least as resistant to tearing, rupturing and disinte¬ gration as is the normal blood vessel of the host animal. Suitable substances that are used to form the grafted artificial material can be derived from synthetic polymers and from proteins, including glycoproteins, that can form matrix structures. Such proteins are often of polymeric type. The matrix structures are weblike with inter- digitating and/or crosshatching strands of protein monomers. Examples of substances which can be used to form the graft materials include Dacron, polytetra- fluorethylene (PTFE) , collagen and fibrin. Mixtures of these substances can also be used, particularly mixtures of collagen and fibrin.

Since it is necessary that a graft of suitable artificial material be placed in an existing blood vessel of the host animal for transudation to occur, a piece of the material is inserted at a selected position in a selected blood vessel. Often, the graft is tubular in structure but such a structure is not required in this invention. If the graft is tubular, it usually will have similar inner and outer diameters as the existing blood vessel into which the graft is inserted. Blood flows through the bore of the tubular graft from one end to the other, originating from and terminating in the blood vessel into which the tubular graft is inserted. If the graft is not tubular, it usually will be in the shape of a patch that covers an opening in the existing blood vessel. The circumference of the patch usually is slightly larger than the circumference of the hole in the existing blood vessel over which the patch is placed. This allows proper securing of the patch graft to the blood vessel. The graft is united with the existing blood vessel such that blood does not leak or escape from the blood vessel at the junction between the blood vessel and the graft material when blood flows through the blood vessel. The graft is positioned so it becomes part of the wall of the blood vessel. The joining of the graft to the existing blood vessel is called inosculation. It is often accomplished with the aid of sutures, such as when surgical anastomosis is performed, but this form of graft attachment to the blood vessel is not required. Any method of inosculation is suitable provided the graft remains in place and blood does not escape from the blood vessel after inosculation as a result of the inosculation. The graft can be inosculated into any existing blood vessel. Preferably, it is inosculated into an artery. The considerations for the size of the graft are that it is to be inosculated into an existing blood vessel and that enough transudation is able to occur through the graft to form a suitable reservoir of transudate after the graft is in place. The amount of transudation that occurs in a given time period is directly proportional to the surface area of the grafted material exposed to the bloodstream. Since the transudate fluid that forms the fluid reservoir originates from the bloodstream of the host animal, the graft surface area directly prescribes the amount of transudate that collects in the fluid reservoir in a given time period. Larger graft surface areas will provide fluid reservoirs in shorter time periods. Concomitantly, they will provide more transu¬ dation of the fluid containing the designated substance into the bloodstream in a given time period when this mode of carrying the substance into the bloodstream is employed. The fluid reservoir, which contains transudate from the bloodstream, can be located anywhere in the body of the host animal. Usually the fluid reservoir is located outside the blood vessels and outside already existing body organs.

The fluid reservoir will normally be formed in the body by natural processes but it may be composed of synthetic materials and subsequently implanted into the body. When composed of synthetic materials, the fluid reservoir has the properties of allowing the transudate to passively enter and to leave the reservoir with little inhibition. With these properties, the fluid reservoir fulfills its role as a transudate repository. Fluid reservoirs composed of synthetic materials can be located near the artificial material graft or remote from it. The fluid reservoirs that form by natural body processes form in the extravascular region of the body as transudate from the bloodstream pools in a particular location. Usually, this location is contiguous with or, at least, close to the artificial material graft that was placed in an existing blood vessel of the host animal. It is not required that the fluid reservoir be located in proximity to the graft. The fluid reservoir can exist in remote anatomical regions from the graft. However, the fluid reservoir location is usually contiguous with or close to the artificial material graft because the transu¬ date from the bloodstream tends to pool in the closest region to the graft that does not resist such pooling and formation of a fluid reservoir. The anatomical boundaries of the naturally formed fluid reservoir are often difficult to precisely locate or define. Since the fluid reservoir forms by a pooling of transudate from the bloodstream, the boundaries of this pool are established by tissues and extracellular sub¬ stances, such as proteins and glycoproteins, that impede the continued movement of the transudate from the graft. As the transudate pooling occurs, extracellular substances are produced or incorporated by the body of the host animal to seal off the fluid reservoir from the other regions of the body. These substances may be continually synthesized or degraded which could cause the anatomical boundaries of the fluid reservoir to shift as time progresses.

The size or volume of the fluid reservoir depends on the amount of transudate held in the reservoir. This amount is controlled by the rate of transudation from the bloodstream, the efficiency of transudate transport from the graft to the reservoir and the rate of loss of the fluid from the fluid reservoir. The loss of fluid occurs by degradation of the fluid constituents, general absorption by the surrounding tissues or seepage from the reservoir boundaries, and transport of the fluid to the bloodstream. Generally, the fluid reservoir is palpable and can be directly observable if a surgical incision to the reservoir site is made. The reservoir can hold between 1 and 250 milliliters (ml) of fluid. Typically, the reservoir is large enough to contain between 10 and 100 ml of fluid.

The structure of the naturally formed fluid reservoir can have a variety of features. It will often have an outer wall composed of cells, such as fibroblasts, and extracellular substances, such as proteins or glycopro- teins, arranged to form a saclike appearance. Such a structure resembles a cyst. In other instances, there may not be an outer membrane but, rather, a dense tangle of tissue with a diffuse outer boundary. The inner region of the fluid reservoir can contain the transudate alone or the transudate including the specified substance and, optionally, other materials such as specified substance carrier molecules. There may be a minor amount of cellular material floating in the fluid. More often, the inner region of the fluid reservoir contains a network made of predominantly protein, glycoprotein or poly- saccharide substances through which the fluid percolates. This network can have a thinly or thickly tangled weblike structure or it can form small cavities or pockets in which the fluid temporarily resides. The texture of this network of proteinaceous material can be lacy or spongy. More often, the latter structure predominates.

A particularly useful and often formed fluid reservoir is a seroma that is formed and located adjacent to the grafted artificial material. The seroma can totally enclose the external surface of the graft, thereby forming a biological barrier between the graft material and normally existing extravascular tissue. The external dimensions of such a naturally occurring seroma that encloses a tubular graft commonly are the length of the graft and 2 to 4 times the diameter of the graft. The volume of such a seroma is approximately 3 to 15 times the volume defined by the length and diameter of the graft. A seroma is composed of transudate on the external surface of the graft surrounded by a dense fibrous, membranous capsule. The transudate is thereby bounded by the external surface of the graft and the fibrous, mem- branous capsule. The seroma as the fluid reservoir is particularly advantageous because it lies contiguous with the artificial material graft and is naturally formed by the body of the host animal in response to the insertion of the graft into an existing blood vessel. When the fluid reservoir is being or has been formed and contains transudate from the bloodstream, the specified substance is incorporated in the fluid for transport to the bloodstream. This incorporation can be achieved by a variety of processes. Many different devices can be used to aid the incorporation of the specified substance into the fluid. In this invention, it is intended that any device or process may be used pro¬ vided the device or process places the specified substance in the fluid without disrupting the transport of transu- date from the bloodstream to the fluid reservoir or transport of the fluid from the fluid reservoir to the bloodstream.

One simple process to place the specified substance into the fluid of the fluid reservoir is by injection or infusion of the specified substance from an extracorporeal depository, i.e. a substance source outside the animal's body, directly into the fluid reservoir. The extra¬ corporeal depository can be any suitable container such as a bottle, bag or syringe. The specified substance is delivered through any suitable conduit such as a tube or syringe needle. This process can be repeated as often as desired to achieve the sought amount or timing of specified substance in the bloodstream of the animal.

Another process to place the specified substance into the fluid of the fluid reservoir is by implanting a repository containing the specified substance in the body of the animal. The respository releases the specified substance for incorporation in the fluid of the fluid reservoir. The implanted repository can be outside or within the fluid reservoir. When outside the fluid reservoir, a process or device must be used to transport the specified substance from the implanted repository to the fluid of the fluid reservoir. For example, a piece of tubing of small diameter can be used to link the implanted repository to the fluid reservoir. This would be satisfactory when the implanted repository is a container, such as a pouch or sealed tube, that contains the specified substance. This arrangement is advantageous when the container must be periodically replaced as the specified substance is utilized or becomes degraded. This arrangement is also useful when the implanted container is programmable to release the specified substance under controlled conditions. The programming feature can be altered without unduly perturbing the rest of the system. It is often preferable that the implanted repository be positioned within the fluid reservoir. This arrange¬ ment provides direct deposit of the specified substance into the fluid of the fluid reservoir without an inter¬ mediating transport system. A container, such as that described above for the implanted repository outside the fluid reservoir, can be used but its placement, and particularly its replacement, may be difficult to achieve.

Other devices are more suitable for implantation within the fluid reservoir. A particularly useful device is a porous network of fibers that essentially fills any space in the fluid reservoir not previously occupied by a solid substance. These fibers can originate from a biological process or be made by a synthetic process. If made by a synthetic process, the fibers must have bio- compatibility properties. This material should not cause an adverse biological reaction in the host animal. Biological substances, including the designated substance, may bind to these fibers but only to the extent that the substances are easily releasable. Cells may adhere to these fibers provided such binding does not inhibit the viability of these cells. In other words, the fibers made by a synthetic process should have the same properties as fibers made by a biological process to be used as an implant device within the fluid reservoir or, even, outside the fluid reservoir. Suitable materials that can form these fibers include polymers formed from proteins, polysaccharides, synthetic substances and mixtures of these substance types.

The specified substance can be embedded in the pores in the porous network of implanted fibers. When implanted within the fluid reservoir, the specified substance is released from the pores as the fluid percolates through the porous network. The specified substance is thereby incorporated in the fluid for subsequent movement to the bloodstream of the animal. The pores of the porous network provide a repository for an adequate quantity of the specified substance in a readily releasable state. An alternative location for the porous network of fibers is in a contiguous relationship with the external surface of the grafted artificial material. For example, when a tubular graft of artificial material is inosculated into an existing blood vessel, the porous network of fibers can be helically, or otherwise, wrapped around the outer surface of the graft. This wrap can be attached before or after the tubular graft is inosculated into the blood vessel. Again, the specified substance can be embedded in the pores in the porous network for subsequent release as fluid percolates through the porous network. The advantage of locating the porous network of fibers on the external surface of the grafted artificial material is that the transudate from the bloodstream incorporates the specified substance from the pores of the porous network as the transudate flows through this porous network. Transudation and specified substance incorporation are united in a common process. The transudate containing the specified substance then continues to the fluid reservoir. When a seroma is the fluid reservoir, the transudate with incorporated specified substance is already in the fluid reservoir when it emerges from the porous network. The combination of seroma and artificial material graft with its contiguous porous network of fibers is an economical use of space and fluid dynamics to provide a fluid reservoir containing the specified substance in trans¬ porting fluid. Another source of the specified substance is bio¬ logical cells that synthesize and secrete the specified substance. These cells can be normally present in the body of the host animal when grafts of artificial material are not inserted. After a graft of artificial material is inserted, the specified substance is produced by these cells and incorporated in the transudate fluid of the fluid reservoir either when it is transudated from the bloodstream or by some other physiological transport process that carries the specified substance to the fluid reservoir. More often, the biological cells are supplied from a source external to the body of the host animal. These cells are implanted devices. They are compatible with the host animal in the sense that they are not rejected by or are protected from the immune system of the host animal and produce the specified substance with the desired properties. They are usually of mammalian origin and often from the species of the host animal. The cells should retain viability and ability to express and secrete the specified substance for a suitable period of time. The cells can be transplanted from another organism or originate from a cell culture. They can be prokaryotic or eukaryotic. They can be transformed or untransformed. They can be aneuploid but usually are diploid.

Particularly useful implanted cells are those arising from recombinant genetic techniques. In these techniques, specified genes encoding the designated substance and, if necessary, nucleic acid sequences involved in the control of expression of the specified genes are inserted into the genome of host cells. After the recombinant cells, with the specified gene recombinantly inserted, are implanted in the host animal, the specified gene is expressed to synthesize the specified substance. The specified substance is subsequently secreted and incorporated in the fluid of the fluid reservoir. The expression of the specified gene is controlled with the aid of the host cell's gene expression control mechanisms.

The recombinantly inserted specified gene can encode for any specified substance that can be produced by genetic expression. Particularly useful substances, for transport into the bloodstream of an animal with this invention, that can be generated by this technique are hormones and factors such as growth factors. The generated hormones include the steroid and polypeptide types. Representative examples of such hormones and factors which can be produced by this technique are insulin, erythropoietin and Factor VIII. The latter factor is involved in the clotting of blood and is lacking in hemophiliacs.

The implanted cells, regardless of their origin, can be located at any of the sites previously described as a locus of the specified substance source. They can be located in containers implanted outside or within the fluid reservoir. These cells can be adherent to the external or internal surface of the walls of the container or suspended in fluid within the container. Alternatively, the implanted cells can be embedded in the porous network of biologically or synthetically made fibers. As previously stated, these fibers can be outside or within the fluid reservoir. The cells can be inter¬ spersed among the fibrous network or adherent to the fibers. When the cells are adherent to the fibers, the bathing media can bring nutrients to the cells and carry away toxic waste products as well as the secreted specified substance. The cells are not removed and lost to the process of producing utilizable specified substance of the invention. When the porous network of fibers is within the fluid reservoir, the cells, whether inter¬ spersed among the network or adherent to the fibers, provide a steady supply of the specified substance ready for transport into the bloodstream of the host animal.

Another suitable location for the implanted cells is adherent to the external surface of the artificial material graft. These cells are continually bathed in the transudate from the bloodstream. The transudate carries nutrients to the adherent cells and waste products and the secreted specified substance away from these cells. The specified substance is then transported in the transudate to the fluid reservoir. When the fluid reservoir is a seroma, the transudate incorporating the specified sub- stance is automatically in the fluid reservoir as it is secreted by the implanted cells. The specified substance is then available for transport into the bloodstream of the animal.

Movement of the specified substance from the fluid reservoir into the bloodstream of the animal is accom¬ plished by one of two processes or a combination of these processes. In one process, the fluid from the fluid reservoir, now incorporating the specified substance, is transudated from the extravascular region of the blood vessel through the grafted artificial material into the lumen of the blood vessel. This is transudation in the direction opposite that which initiated the pooling of fluid in the fluid reservoir. When the fluid reservoir is a seroma, transudation from the extravascular region into the bloodstream can quickly occur since the fluid is contiguous with the external surface of the grafted artificial material. In this instance, simultaneous transudation in both directions usually occurs as fluid moves out of and into the bloodstream through the grafted artificial material.

The other process of moving the fluid incorporating the specified substance into the bloodstream of the animal is by drainage from the fluid reservoir through the lymphatic system into the bloodstream. The lymphatic system includes lymphatic vessels which carry fluid from extravascular and extracellular regions of the body to thoracic and lymphatic ducts which, in turn, empty into the subclavian veins of the bloodstream. Often, lymphatic vessels form and, if possible, infiltrate abnormal structures in the body as a reaction to the presence of these structures. For example, lymphatic vessels are usually observed intermeshed with the tissues of a seroma. The lymphatic vessels drain extracellular fluid from these structures. Fluid containing the specified substance thereby moves from the fluid reservoir, which is such an abnormal structure, into the lymphatic vessels where it is transported via the thoracic or lymphatic duct to the bloodstream. The specified substance is then transported throughout the body of the host animal and can react with the desired target tissue. In addition to methods of introducing a specified substance into the bloodstream of an animal from a particular type of fluid reservoir located in the recipient animal's body, this invention also relates to the assemblage necessary for such specified substance introduction into the bloodstream. The assemblage in¬ cludes the blood vessel graft of artificial material that allows transudation to occur from the blood stream to the extravascular space, the fluid reservoir formed to contain this transudate and a source of the specified substance. The artificial material that forms the graft can be composed of any substance or substances that allow transu¬ dation to occur between the bloodstream of the host animal and the extravascular space. These substances include artificial polymers and protein, including glycoprotein, matrices. Examples of such polymeric substances include Dacron, polytetrafluroethylene (PTFE) , polyurethane, collagen, fibrin and mixtures of these specific sub¬ stances. The polymer material can be fabricated with pore sizes ranging from microporous (1 to 10 micron diameter) to more commonly employed pore sizes of 30 to 90 micron diameter to macroporous knitted fibers. When the latter porous construction is used, reliance is placed on blood coagulation to partially seal the interstices between the polymeric strands.

The fluid reservoir is extravascular and usually outside any existing organ cavity. The fluid reservoir is formed in the body of the host animal to contain the transudate that crosses the grafted artificial material from the bloodstream to the extravascular region outside the graft. This reservoir can be in a region of the body remote from the graft or, more likely, close to the graft. Fluid reservoirs close to the graft include those reservoirs formed in the perigraft region of the extra¬ vascular space surrounding the graft that is contiguous with the graft and those reservoirs slightly removed from the graft. A particularly useful fluid reservoir is a seroma which forms adjacent to the grafted artificial material. The fluid reservoir of this invention can be void of any solid material in its internal regions or it can contain a network of porous fibers. These fibers can be composed of polymers from natural or synthetic sources such as proteins, glycoproteins, polysaccharides, synthetic polymers and mixtures of these specific sub¬ stances. The network can be tenuous or dense.

The fibers of the porous network can be bare or they can have cells adhering to them. In other instances, the biological cells can be adherent to the external surface of the artificial material graft. These cells can be prokaryotic or eukaryotic. These cells synthesize and secrete the specified substance into their environment which is the transudate occupying the fluid portion of the reservoir. Although these cells can be nonrecombinant, they often are formed by recombinant techniques in order to express the specified substance from an inserted gene. With this technique, the specified substance and host cell can be controlled to yield a stable source with few, if any, adverse properties. Particularly important substances expressed from recombinantly inserted genes are polypeptide hormones, steroid hormones and factors. Examples of these substances are insulin, erythropoietin and Factor VIII.

The invention is further illustrated by the following examples. These examples are not intended to be limiting of the invention in any way.

EXAMPLE I

INSERTION OF BLOOD VESSEL GRAFT, TRANSUDATION

AND FORMATION OF SEROMA

MATERIALS AND EXPERIMENTAL METHODS

Animal care complied with the "Principles of Laboratory Animal Care" (formulated by the National Society for Medical Research) and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, Revised 1985) . Eight knitted single velour Dacron grafts, 50 cm long, 8 mm diameter, (C.R. Bard, Inc. , Billerica, Massachusetts) were individually placed in eight large (> 25 kg) dogs. The common carotid artery was divided and an end-to-end anastomosis was constructed between the mobilized proximal common carotid artery and the inflow end of the Dacron graft. The graft was then brought through a subcutaneous tunnel dorsal to the left shoulder and around the flank to the distal aorta which was exposed through a retroperitoneal approach. An anastomosis was constructed between the distal end of the Dacron graft and the side of the terminal aorta. The aorta was ligated just proximal to this anastomosis. Flow rates were measured in the grafts using an ultrasonic (Transonics, Inc., Ithaca, New York) flow probe. Blood samples were taken from the graft simultaneously at sites adjacent to the proximal and distal anastomoses at various time intervals from 1 hour to 9 months following implantation of the graft. A 19 gauge butterfly was used to collect samples into EDTA, 12% w/v. The rate of blood aspiration was timed and standardized at 10 cc/10 sec. A Model S880 Coulter Counter was used to determine red blood cell count (RBC) and mean corpuscular volume (MCV) . All analyses were performed by an independent laboratory following a blinded sample analysis procedure. The hematocrit was determined as the follows:

RBC x MCV sample volume

Manual spun hematocrits were randomly performed to verify Coulter Counter derived hematocrits.

After eight months, the dogs were anesthetized, and grafts were exposed proximally and distally. 5,000 U of heparin was infused intravenously. A 16 gauge butterfly catheter was placed in the native vessel proximally and distally. Inflow and outflow through the graft was occluded with vascular clamps, and the dogs were sacri¬ ficed with saturated KC1. The grafts were immediately infused with normal saline until the effluent was clear. The grafts were then perfused with 2% glutaraldehyde, 4% formaldehyde in phosphate buffered saline at 100 mm Hg pressure from a pressure bag for 15 minutes. The distal butterfly was clamped and then the proximal line was clamped so that the pressure in the graft remained at 100 mm Hg. The graft was then exposed throughout its length and removed, along with the perigraft tissue. Transverse and longitudinal sections were cut from the midportion of the graft and critical point dried. The sections were then sputter coated with gold/palladium (60/40) . Scanning electron microscopy was performed.

STATISTICAL METHODS

The differences in results from the proximal to distal sample sites drawn simultaneously at the same time point were evaluated by a general linear regression model (SAS release 6.03, SAS Institute Inc., Cary, North Carolina) . The model for each analyte at the proximal site included the analyte baseline for the proximal site, the measurement time, and the distal site for comparison. Each dog was included as a random event. This regression model allowed for the testing of the effect of time and was also used to establish a linear relationship between the two sample sites.

RESULTS To ensure that the hematocrit differences were.not artifactually produced on the Coulter Counter, manual spun hematocrits were randomly compared.

This comparison demonstrated excellent correlation of results and no evidence of artifacts influencing the Coulter results.

Up to 48 hours following implantation of the graft, there was no hematocrit difference between the proximal and distal samples. Without exception, in all subsequent sample pairs, the distal hematocrit was greater than the proximal. This difference was first noted in the 72 hour samples. Prior to the 72 hour time, there was no signifi¬ cant change (0.24 +/- 0.29, p < 0.4205) ; for all data points after 72 hours, there was a significant (2.46 +/- 0.28, p = 0.0001) increase in HCT across the graft. The hematocrit increase was fully accounted for by the in¬ crease in RBC. There was no change at any time in MCV across the graft. There was a near perfect correlation between the hematocrit increase and the RBC increase. On the other hand, there was virtually no correlation between hematocrit increase and MCV. Average flow rate through the grafts was 189 +/- 49 cc/min.

The hemoconcentration described in the above data demonstrates a net transmural fluid loss from the graft lumen. This can be calculated as follows:

Area of graft (uncrimped) = x x DIAMETER x LENGTH = 3.14 X 0.8 x 50 = 126 cm²

Rate of fluid loss = (HCT DISTAL - HCT PROXIMAL) x FLOW RATE = 0.0246 x 189 = 4.6 cc/min.

Average rate of fluid loss/area of graft surface = 4.6/126 = 0.036 cc/cm /min

When examined histologically, all grafts were sur¬ rounded by a dense, cellular, adherent capsule (see Figure) . This capsule enclosed a region external to the graft devoid of cellular or fibrous material. EXAMPLE II MEASUREMENT OF TRANSUDATION BY COLLECTION OF TRANSUDATE

Animals, materials and surgical procedures were the same as in Example I. In this Example, a Dacron graft was surgically exposed one month after implantation. A 3 cm segment of the graft was isolated and wrapped with a polyethylene sheet. The surgical exposure was then closed. After 24 hours, the graft was exposed again and the fluid that had collected between the polyethylene sheet and the Dacron graft was collected and measured.

The amount of fluid collected was approximately 3 cc.

Area of graft covered by polyethylene sheet = x DIAMETER x LENGTH - 3.14 x 0.8 x 3 = 7.5 cm²

Amount of fluid loss/area of graft surface = 3/7.5 = 0.4 cc/cm²

Average rate of fluid loss/area of graft surface = 0.4/1440 = 2.8 x 10~⁴ cc/cm²/min

Although this fluid loss rate is much less than that determined in Example I, it represents significant transu¬ dation and is adequate for supplying an extravascular reservoir of transudate fluid. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

1. Use of an artificial vascular graft for the formation of an extravascular reservoir for use in the delivery of a substance into the bloodstream, whereby: a) an artificial graft is inosculated into a blood vessel of said animal and transudation occurs through said artificial graft from the bloodstream to an extravascular space, thereby forming an extravascular reservoir; b) said substance is introduced into the body of said animal under conditions appropriate for said substance and the transudate fluid to combine, thereby producing a combination of said substance and said transudate fluid, and c) said combination is maintained under conditions appropriate for said combination to be transported to the bloodstream of said animal.

2. The use of Claim 1 wherein said substance is from an extracorporeal depository.

3. The use of Claim 1 wherein said substance is from a source selected from the group consisting of a porous, fibrous network of biocompatible material residing within said extravascular reservoir, cells that express and secrete said substance, and an implanted repository containing said substance. 4. The use of Claim 3 wherein said cells are recombinant.

5. The use of Claim 3 wherein said biological cells are adherent to the external surface of said artificial graft.

6. The use of Claim 5 wherein said cells are recombinant.

7. The use of Claim 3 wherein said porous, fibrous network of biocompatible material is formed from a polymeric material selected from the group consisting of proteins, polysaccharides, synthetic substances and mixtures thereof.

8. The use of Claim 7 wherein said substance is releasably lodged within the pores of said porous, fibrous network or is expressed and secreted from viable cells adhering to said porous, fibrous network.

9. The use of Claim 8 wherein said cells are recombinant.

10. The use of Claim 7 wherein said porous, fibrous network of biocompatible material is adherent to said artificial graft.

11. The use of Claim 1 wherein said artificial graft is fabricated from a material selected from the group consisting of synthetic polymers and protein matrices. 12. The use of Claim 11 wherein said material is further selected from the group consisting of Dacron, polytetrafluoroethylene, polyurethane, collagen, fibrin, and a mixture of collagen and fibrin.

13. The use of Claim 4 wherein said cells express and secrete said substance which is selected from the group consisting of a polypeptide hormone, a steroid hormone and a biological factor.

14. The use of Claim 13 wherein said substance is selected from the group consisting of insulin, erythropoietin and Factor VIII.

15. The use of Claim 1 wherein said combination is transported to the bloodstream of said animal via a pathway selected from the group consisting of drainage through the lymphatic system, transudation through said artificial graft from the extravascular side of said artificial graft to said bloodstream, and the combination thereof.

16. The use of Claim 1 wherein said substance is introduced into a seroma lying external and adjacent to said artificial graft. 17. An assemblage for introducing a substance into the bloodstream of an animal, comprising: a) an artificial graft inosculated into an existing blood vessel of said animal, wherein said artificial graft is fabricated of a material through which transudation can occur through said artificial graft between the bloodstream and an extra¬ vascular space; b) an extravascular reservoir of transudate fluid, wherein said extravascular reservoir is formed in said extravascular space and said transudate fluid is formed by said transudation that occurs from the blood- stream to said extravascular space; and c) a source of said substance, wherein said source can provide said substance directly into said transudate fluid in said extra¬ vascular reservoir.

18. The assemblage of Claim 17 wherein said artificial graft is fabricated from a material selected from the group consisting of synthetic polymers and protein matrices.

19. The assemblage of Claim 18 wherein said material is further selected from the group consisting of Dacron, polytetrafluoroethylene, polyurethane, collagen, fibrin and a mixture of collagen and fibrin. 20. The assemblage of Claim 17 wherein said extra¬ vascular reservoir is selected from the group consisting of an extravascular space in the perigraft region of said artificial graft, a seroma formed in the perigraft region of said artificial graft, and a remote extravascular space in the host animal's body.

21. The assemblage of Claim 20 wherein said extra¬ vascular reservoir is infiltrated with a porous, fibrous network comprising a polymeric material selected from the group consisting of proteins, polysaccharides, synthetic substances and mixtures thereof.

22. The assemblage of Claim 21 wherein cells that express and secrete said substance are adherent on said porous, fibrous network.

23. The assemblage of Claim 22 wherein said cells are recombinant.

24. The assemblage of Claim 23 wherein said sub- stance is selected from the group consisting of a polypeptide hormone, a steroid hormone and a biological factor.

25. The assemblage of Claim 24 wherein said sub¬ stance is further selected from the group consisting of insulin, erythropoietin and Factor VIII. 26. The assemblage of Claim 18 wherein cells that express and secrete said substance are adherent on the external surface of said graft material.

27. The assemblage of Claim 26 wherein said cells 05 are recombinant.

28. The assemblage of Claim 27 wherein said sub¬ stance is selected from the group consisting of a polypeptide hormone, a steroid hormone and a biological factor.

1029. The assemblage of Claim 28 wherein said sub¬ stance is further selected from the group consisting of insulin, erythropoietin and Factor VIII.