WO1991012820A1

WO1991012820A1 - Low affinity drugs

Info

Publication number: WO1991012820A1
Application number: PCT/SE1991/000128
Authority: WO
Inventors: Sten Ohlson
Original assignee: Sten Ohlson
Priority date: 1990-02-28
Filing date: 1991-02-21
Publication date: 1991-09-05
Also published as: SE468238B; SE9000725D0; SE9000725L

Abstract

The invention relates to a new way for guiding a pharmaceutical to specific targets such as microorganisms as bacteria and viruses, tissue cells or tumor cells. The mechanism of targeting is characterized by a number of weakly interacting sites - Kass < ca 104 M-1 - which by cooperation can realize a strong and specific binding to the target which can be e.g. surface antigens on bacteria, viruses or tumor cells. Alternatively the target can be structures of the host cell which can interact with the foreign substance. This weak affinity pharmaceutical can act in different ways by preventing and blocking the binding of the microorganism/tumor cell to its target, host cell or by an improved targeting of the pharmaceutical to the specific binding place. The pharmaceutical system is generally composed of a number of weak affinity sites that is defined and with a specific configuration that a binding to the target is achieved. The combination of affinity sites is obtained by various carriers such as microparticulate substances, macromolecules and low molecular weight substances. In addition, substances are included such as cell poisons, which are used to destroy the invading microorganism/tumor cell.

Description

"LOW AFFINITY DRUGS"

The invention relates to pharmaceuticals with weak affinity. According to the invention the pharmaceutical is characterized by a number of, on a carrier in terms of a particle or a mole¬ cule, bound, preferably covalently, weak affinity sites for binding and neutralizing virus, bacteria, tumor cells or other pathogens by multiple binding. The multiple binding can be simultaneous .

The human being is daily exposed to agents all from microorga¬ nisms to basic chemicals, of which many of these exposures will have a negative effect and will cause infections with possible disease as a result. The structures of the human body such as its cells can be altered and converted to foreign configurations which can cause serious imbalances in the biological function. One example is the transformation of normal cells to tumor cells which grow uncontrolled and penetrate various parts of the human body.

A number of different, microorganisms - such as bacteria, virus, rickettsiae and fungi and their toxins - can, if penetration is allowed, give rise to infectional diseases and toxin poisoning.

Bacteria are unicellular microscopic organisms (usually 0.5-5 |lm in size) . There are about 1500 known species of which many are pathogenic to mankind, and new species are continually disco¬ vered. Many of these bacterial infections can be treated with medicines.

Viruses are very small parasites or pathogens (10-300 n in size) which only can grow in living cells. Vaccination is a powerful tool to protect yourself from virus infection. However, against some virus there are no efficient vaccination. Viruses have the capacity to convert into shapes, not easily recognized by vaccination. There are no efficient procedures or medicines available against these kind of viruses.

SUBSTITUTE SHEET Rickettsiae are intracellular parasites present in various in¬ sects. They are less in size than bacteria and is morphologi¬ cally not as well-defined as bacteria.

Fungi are plants without chlorophyll. They are usually of multi- cellular nature but are not well differentiated. They vary in size from unicellular to large multicellular structures.

The invention will be described in detail as it relates to bac- teria, virus and tumor cells but it can be applied as well to other microorganisms and to toxins where applicable.

Every bacterium, virus or tumor cell have an outer surface which is unique and constitutes an important unity for communication and protection against the surrounding environment. This outer limitation in terms of a cell- or capsid wall has a complicated structure of proteins, lipids and carbohydrates, and this mosaic of molecules forms integrated units with defined function. For example, there are receptors for different interacting molecules such as hormons and other special molecules that can bind to the target cells.

The structure of the outer surface identifies the biological unit and the presence of foreign surface antigens can lead to an immunological (humoral- or cell mediated) reaction at the host with the purpose to eliminate the intruding agent; viruses, tumor cells or other microorganisms. There are a number of mechanisms for elimination such as complement activation, phagocytosis with the aid of macrophages and granulocytes, action from lymphokines e.g. interferons and interleukins, attacks by cytotoxic T-cells and K-cells and direct action from blocking antibodies.

As a central theme in the research activities of the pharmaceu- tical industry for establishing efficient therapeutical proce¬ dures, the aim has been to design pharmaceuticals that in different ways interfere with the pathogenic agent and their interaction with the target or alternatively how the pharmaceu- tical interacts with the target structure on the cell/capsid surface. This interaction has to be as specific as possible without affecting the surroundings, i.e. the host in a negative manner and create a biological imbalance.

As an illustrative example on specificity versus the host is the use of classical antibiotics such as penicillin which prevent the synthesis of the cellular wall of many types of bacteria in a way that effectively stops the growth of the bacteria popula- tion without having any detrimental effects on the biological activity of the host.

The modern pharmaceutical industry, especially the pharmaceuti¬ cal biotechnology industry which utilizes the latest achieve- ments in recombinant DNA- and hybridoma technology, is manufac¬ turing interacting substances usually of macromolecular nature e.g. monoclonal antibodies. These 'biological' pharmaceuticals are characterized by a strong binding to their target, where the interacting structures are reversibly binding to each other under high affinity; the binding constant Kass > 10⁵ M"¹. Another approach is to employ rational drug design and make small analo¬ gues of the 'biological' pharmaceutical with the same characte¬ ristics as the acromolecules described above.

However, these 'high-affinity' pharmaceuticals have a number of serious problems: Due to their high binding strength to the tar¬ get, they often bind to irrelevant structures outside the tar¬ get. An important 'protective' mechanism for certain pathogenic agents and tumor forms is that their surface structures (surface antigens) are released in significant amounts as a sort of decoy molecules . Certain carriers of tumors often have circulating complexes of tumor antigen -antibody and these complexes have been able to block the cytotoxic effect of T-cells as well as K- cells.

As a result of this smoke screen of decoy molecules the 'high- affinity' pharmaceutical cannot reach the relevant surface structure of the pathogen/host and no therapeutic effect is gained . In other instances, the goal can be to differentiate between an infectious agent/ tumor cells with high density of surface antigens and host structures with limited presence of antigen , and again there is a situation where the 'high-affi- nity' system has difficulties to distinguish between friend and enemy and the effect of treatment is inadequate.

The invention relates to a new design of pharmaceuticals, where molecular structures are weakly interacting with its target, and it is below described in more detail with several examples of possible application areas, without being limited to these exam¬ ples.

In the following, the concept 'ligand' is used for molecular configurations which are immobilized on cell/capsid surfaces of the pathogen/tumor cell and the concept 'ligate' for ligand- interacting, soluble alternatively bound structural elements on the host's cellular surfaces.

The purpose of the invention is to create a pharmaceutical sys¬ tem, where its target-guidance mechanism is characterized by a number of weakly interacting sites that by weak individual affi¬ nity interacts with its target. The target can be ligand struc¬ tures of type surface antigens on viruses and bacteria. Alterna- tively, tumor cells or ligate structures of the host can also be the target. Weak interaction^' is defined as binding strength of Kass < approx. 10⁴ M^_1. A sufficient specificity to the target structure is achieved by cooperation by several weakly interac¬ ting sites of the pharmaceutical system which together bind the target with adequate strength. In other words the desirable spe¬ cific binding is realized by molecular cooperativity which gives the necessary avidity for a strong and specific binding of the target structure. If, for example, the binding strength of the individual recognition site of the pharmaceutical system is K_ass = 10² M^-1, which as such is too weak to produce a rele¬ vant binding . By cooperative binding of e.g. three sites, a total avidity of K_ass = 10⁶ M^-1 can be achieved which should be sufficient for a strong anchoring to the target. In this way many of the problems can be avoided that relate to traditional pharmaceutical targeting with high-affinity monoclo¬ nal antibodies. Examples of problems with traditional pharmaceu- tical targeting where the new weak affinity concept can be applied to:

- Ligand structures of e.g. antigens on bacteria, viruses and tumor cells which can be present in varying concentrations and only is distinguished from the host by their numbers. As a result there can be too high specificity of the high affinity pharmaceutical which then miss the target and bind non-desirably to the host.

- Release of circulating ligand of type antigens from invading agents which then can inactivate the high-affinity pharmaceuti- cal by too strong individual binding.

- In the case of low substitution of ligand at e.g. the cell surface, a traditional high-affinity pharmaceutical of e.g. a monoclonal antibody with an attached cytostatic agent can be insufficient to cause an adequate effect.

When studying the natural interaction existing in biological systems you find that it is often weak in it's nature. Thereby the dynamics of the system is guaranteed and biological effects may be directly dependant of changes in the surroundings. An example of this is the interactions of different enzymes with their substrates and inhibitors, where the activity of the enzyme is directly dependant of changes in concentration of interacting substances.

The achieved specificity of recognition in biological systems is often the result of molecular cooperativity of individual weak interactions which jointly give the desired response. A representative example is the by Clq in complement activation. The role of the complement is to destroy hostile agents such as bacteria, tumor cells or fragments thereof. The first complement factor; Clq do bind to the hostile entity by recognizing anti¬ bodies that are aggregated or linked together onthe foreign cell surface. Clq is a large protein with six globular subunits which individually bind weakly (K_aSs ⁼ 10³ M^-1) to the antibody. A strong and specific binding of Clq to the aggregate of antibo¬ dies, e.g. bacteria covered with antibodies is realized by the cooperation of several of the binding sites of the Clq molecule. This is the fact which enables Clq to differentiate between monomeric and polymeric immunoglobulin by cooperativity of weak binding events.

Another example is the classical agglutination reaction, where frequently Ig (a pentamer with ten binding sites) interacts with weak individual binding and the specificity is then the result from the simultaneous use of several binding sites.

Similar mechanisms can be observed when studying cell-to-cell interactions e.g. the synthesis of specific types of tissue, where multiple weak binding frequently mediated by special adhe¬ rent molecules form the basis for recognition and differentia¬ tion.

As exemplified above there are many natural- systems that operate according to the principles of the invention. Therefore an extensive amount of material support the theory behind the invention.

The principle to use weak affinity for various chromatographic operations has been exploited before - Ohlson, Swedish patent no. 8700949-0 - where structurally similar substances were sepa¬ rated and analyzed with the use of weakly interacting monoclonal antibodies. It has been shown that weak binding - K_ass = 10²-10⁴ M^-1 - can be used in chromatographic systems, where adequate selectivity and retention can be realized by a repeated number of weak, but slightly different interactions between the immobi¬ lized interacting substance on the solid phase and solutes in the mobile phase.

A pharmaceutical system according to the invention is achieved by a number of weakly interacting sites, weak affinity sites acting as target-guided units which individually do not bind its target with sufficient strength but in cooperation the desired specificity is achieved and as a result the anchoring onto the target is complete.

These weak affinity sites are bound to a carrier in such amount and with such a configuration that optimal selectivity is achie¬ ved. The cooperation can occur with at least 2, often 3-6 affi¬ nity sites in order to achieve the desired effect. Microparticu- late substances, macromolecules and defined low molecular weight substances can be used as potential carriers. A number of natu¬ ral and artificial particles can also be used as microparticu- late carriers. Colloidal systems (size of 5-1000 nm) are also suitable as carriers. The carriers can be composed of e.g. phospholipids (liposo es) , polysaccharides such as cellulose, gelatin, polyacrylamide and proteins such as denatured albumin and cellular structures such as subcellular particles. Soluble macromolecules can also be used as effective carriers for weak affinity sites. Examples from this category are numerous: Plasmaproteins such as lipoproteins, nucleic acids such as DNA, polysaccharides such as dextran, glycoproteins and

(semi) synthetic polymers such as polyethyleneglycol and polyami- noacids. Furthermore, low molecular weight substances can be used such as defined peptide- and oligosaccharide sequences and organic molecules with handles for weak affinity sites .

According to the invention a weak affinity site is a defined molecular structure that interacts weakly (see above) with its target . These weak affinity structures can be all from a small structural element to large complicated molecules.

The use of monoclonal antibodies is presently an established technology within such areas as imaging, diagnostics and thera¬ peutical products. Weak monoclonal antibodies can according to the invention be utilized as weak affinity sites . In addition, the antibody can be split into its parts; F_ab, heavy- and light chains and single domain units which all can be weak affinity molecules. In this connection recombinant single-chain antibo¬ dies can be of interest. Furthermore, short peptide sequences (5-50 amino acids) are potential candidates for the weak affinity technology as they in some cases resemble parts of the hypervariable regions of the intact antibody.

Another approach is to weaken (recombinant) protein- and peptid molecules with well-defined high affinity function by gene mani pulation e.g. site-directed mutagenesis.

Furthermore, low molecular weight receptor analogues can be important weak affinity structures which interfere and prevent binding of e.g. virus to its target cell. Similarly, short carbohydrate sequences with weak affinity can bind its target through lectins at the cell surface. Synthetic substances which fulfil the conditions for weak affinity can also be used as an alternative to the biological systems described above.

When dealing with extremely weak binding - K_ass < ca 1 M"¹ - at the interacting site, only non-specific effects can be seen and as such this binding is not part of the technology as described in the invention.

In other words individual ionic- and hydrophobic groups can not produce the result as described by the invention. However, in some cases the desirable' weak affinity effect can be achieved according to the invention when K_ass = ca 0.5 M"¹.

Finally, the carrier can incorporate, if necessary, the agents required to eliminate the hostile cell/virus with such tools as cellular poisons (toxins), enzymes and radioactive isotopes. In other connections e.g. at virus therapy blocking of the trans¬ port to the host cell can be sufficient to produce a therapeuti¬ cal effect.

The immobilization technology as applied according to the invention is intended to effectively bind the selected substance - the weak affinity structure or other substance - covalently or not and with maximal maintained function. Various methods for immobilization is described e.g. in Methods in Enzymology Vol. XLIV, 1976, K. Mosbach editor, Academic Press, New York.

In the design of specific pharmaceuticals the way to administer the pharmaceutical has to be taken into consideration. Different procedures are here available such as intravenous, oral, subcu¬ taneous and transdermal. The most effective way is usually to take advantage of the natural transport system namely the vascu¬ lar system. If the goal is to reach an extra-vascular effect the pharmaceutical system has to pass certain biological barriers. The vascular capillary system is surrounded by several barriers such as endothelial cells, basal membranes and last but not least the reticuloendothelial (RES)- and macrophage system which act as an effective block for unfamiliar substances. According to the invention the above described weak affinity pharmaceuti- cal system has as it relates to the procedures for administra¬ tion of pharmaceuticals the same potential and restrictions as a more traditional pharmaceutical-delivery system.

In the following some application areas for the invention, but not limited to, will be described in some detail:

AIDS is a very serious immunodeficiency syndrome with no available effective method of treatment. A specific virus- commonly named the HIV virus-attacks and breaks down the immune defense of the human body where the white T-cells are its main target. It has been possible to identify the molecular assembly (CD4) which define the T-cell receptor, where the HIV-virus attaches to before penetrating the cells. It has been suggested that a possible therapy is to prevent the virus to reach its target cell by providing a pharmaceutical that either binds to the receptor on the cell surface or binds and neutralizes the virus before it reaches its target.

The research efforts in this area have been concentrated towards establishing a CD4 therapy where artificial soluble receptor (CD4) is injected and act as a decoy and 'suck up' the virus before entering the target cell. The preliminary results obtained so far indicate a positive effect, but the CD4 is too short-lived and it is hard to maintain the amounts necessary to prevent infection. Another possible route is to use gene therapy.

Another complication is to attain sufficient specificity by the action of the pharmaceutical. The HIV-virus releases a number of different molecules e. g. surface antigens as gp 120. The gp 120 is responsible for the binding to the CD4-receptor. When the HIV virus is eliminated, the body's lymphatic- and vascular system will be loaded with free virus antigens which will substantially aggravate any therapeutical effect.

According to the invention e.g. small fragments of CD4; peptides or similar substances with defined weak affinity can be produced and by a suitable carrier construction they can cooperate and achieve a cooperative binding to the virus surface. By this procedure as suggested by the invention the problems with the non-specificity and rapid inactivation of the present technology can be avoided. In general terms by fragmentation of the CD4 molecule ligand analogues are produced that are weakly binding and the challenge is then how to use these efficiently. In accordance with the above ligand analogues such as monoclonal antibodies or fragments thereof can be interesting alternatives characterized by weak and individual binding and by effective cooperation they can bind and block the CD4 receptor on the T- cell surface. This weak affinity pharmaceutical system can natu¬ rally be supplied with all the arsenal of anti-viral substances that is required to receive intended destructive effect on the virus population.

Another area of application for the weak affinity technology according to the invention is the potential for cancer therapy by using ligand analogues or other recognition units such as antibodies or fragments thereof again characterized by weak interaction to its target. By cooperative action of several weak affinity sites a strong and specific binding of the pharmaceuti¬ cal system to the tumor cell is achieved with a subsequent action of e.g. various cell poisons. For tumors treatable with immunological means the weak affinity pharmaceuticals can be of importance while others with no or little antigenicity cannot be controlled in its growth by the weak affinity approach.

Another area that illustrates the potential of the invention is the possibilities to use weakly interacting oligosaccharide sequences to guide pharmaceuticals to carbohydrate-binding pro¬ teins of type lectins on the surface of the cell or in the cell membrane. Weak binding sites can be put together in the polysac- charide that effectively bind to the target e.g. bacteria-speci¬ fic lectins. In this manner the adherence of bacteria to the cell surface of the host can be prevented or alternatively they can attack and destroy the invading bacteria with an array of bacteria poison.

The use of the weak affinity interactions according to the above described technology is a new way for a rational design of pharmaceuticals according to biological principles.

The technology is expected to have significant importance for the combate against infectional diseases and for certain tumor forms.

If this technology can lead to a breakthrough for e.g. AIDS- research it will have a tremendous effect on the society. The weak affinity concept according to the invention is also expected to have applications in other areas of importance.

Claims

C L A I M S

1. A pharmaceutical, characterized by a number on a carrier in terms of a particle or a molecule of preferably cova- lently bound, weak affinity sites, where the individual weak af¬ finity for every binding site is approximately 1 < K_ass <10⁴ M^-1, for binding and neutralizing virus, bacteria tumor cells or other etiological agents by multiple binding.

2. A pharmaceutical according to claim 1, characterized the fact that- the multiple binding is simultaneous.

3. A pharmaceutical according to claim 1 or 2, characteri¬ z d by the fact that the weak affinity sites are composed of antibodies or fragments thereof, recombinant single chain anti¬ bodies, short peptide- or saccharide sequences, synthetic sub¬ stances or similar entities.

4. A pharmaceutical according to any of above claims, charac- terized by the fact that the carrier is composed of micro- particles, preferably colloidal systems and/or macromolecules with sizes of 5-1000 nm.

5. A pharmaceutical according to any of above claims, charac- terized by the fact that the carrier is composed of phos- pholipids, polysaccharides, proteins, and/or cellular structu¬ res such as native cells.

6. A pharmaceutical according to claim 4, characterized by the fact that the carrier is composed of soluble macromole¬ cules, such as plasmaproteins, nucleic acids, (semi)synthetic polymers, organic molecules with binding areas for weak affi¬ nity.

7. A pharmaceutical according to any of above claims, charac¬ terized by the fact that the carrier contains cell poison, enzymes or radioactive isotopes, or that the carrier is composed of the above mentioned substances.

8. The use of the pharmaceutical according to any of above claims, characterized by the fact that the administration of pharmaceutical is intravenous, oral, subcutaneous, transder- mal or similar.

9. The use of the pharmaceutical according to any of above claims, characterized by the fact that the pharmaceutical is blocking the interaction between the cells and the disease- causing agent.