WO1995034811A1

WO1995034811A1 - Protein condensation inhibitors and methods related thereto

Info

Publication number: WO1995034811A1
Application number: PCT/US1994/006575
Authority: WO
Inventors: George B. Benedek; George M. Thurston; John I. Clark
Original assignee: Oculon Corporation; Massachusetts Institute Of Technology
Priority date: 1993-06-11
Filing date: 1994-06-10
Publication date: 1995-12-21
Also published as: AU7104994A

Abstract

There are disclosed protein condensation inhibitors which are useful in the treatment of phase transition disease states associated with the phase transition of proteins from a soluble to a condensed state. Methods of identifying compounds for their ability to function as protein condensation inhibitors, as well as the use of such protein condensation inhibitors to treat phase transition disease states, are also disclosed. In one embodiment, a method of identifying compounds comprises assaying the compound in an aqueous protein solution to determine whether the sign of δE is positive. In this method, the assaying may include the steps of measuring a change in phase separation temperature and determining whether protein condensation occurs upon raising or lowering the temperature of the aqueous protein solution. Preferably, the aqueous protein solution comprises a protein associated with a phase transition disease state, such as sickle hemoglobin. In another embodiment, a method of identifying compounds involves assaying the compound in at least two distinct aqueous protein solutions for its ability to prevent or delay a temperature dependent phase transition to a condensed state in each of the distinct aqueous protein solutions. Examples of suitable proteins for the distinct aqueous protein solutions include lysozyme and sickle hemoglobin.

Description

PROTEIN CONDENSATION INHIBITORS AND METHODS RELATED THERETO

Technical Field

This invention is generally directed to protein condensation inhibitors and, more specifically, to methods of identifying protein condensation inhibitors and the use thereof to treat phase transition disease states.

Background of the Invention

The understanding of human disease has evolved over many decades. During that time, two general paradigms for human disease have emerged: first, that bacterial and viral agents are responsible for infectious disease; and, second, that disease may be genetic in origin. The first paradigm led to the development of antibiotics to block growth and development of bacteria, as well as to the development of vaccines to stimulate the immune response against viral infection. The second, and more recent paradigm, followed the recognition that errors in gene coding may produce corresponding proteins which are defective in both sequence and structure. Using this second paradigm for human disease, progress has been made in identifying individual genes or groups of genes responsible for specific metabolic diseases.

Although great strides have been made in understanding human disease, many diseases exist which can not be readily explained by the above paradigms or, if understood, cannot yet be treated. Accordingly, there is a need in the art for agents and methods to treat such diseases. The present invention fulfills this need, and provides further related advantages.

Summary of the Invention

In brief, a new paradigm of human disease is disclosed which, prior to this invention, has not been formally recognized. A critical aspect of this new class of disease (referred to herein as "phase transition disease states") is that specific proteins which are normally soluble undergo a phase transition to a condensed state. As used herein, a "condensed state" includes an amorphous or crystalline solid, a gel, a condensed liquid phase, a condensed liquid crystalline phase, or a distribution of protein clusters, aggregates or polymers of various size. The presence of the condensed state results in the damage to cellular and/or organ function associated with a particular phase transition disease state.

In the practice of the present invention, a protein condensation inhibitor

("PCI") is disclosed for use in the treatment of a phase transition disease state. A PCI of this invention functions by either shifting the location of the phase boundaries for a specific protein associated with the phase transition disease state, or by affecting the kinetics of phase transition from the soluble protein to the condensed state.

In one aspect of the present invention, there is disclosed a method of identifying a compound which functions as a protein condensation inhibitor by assaying the compound in an aqueous protein solution to determine whether the sign of δE is positive. Such assaying may include the steps of measuring a change in phase separation temperature of the aqueous protein solution by the addition of the compound, and determining whether protein condensation of the aqueous protein solution occurs upon raising or lowering its temperature. In a preferred embodiment, the aqueous protein solution comprises a protein associated with a phase transition disease state, such as sickle hemoglobin.

In another aspect of the present invention, a further method of identifying a compound for its ability to function as an PCI is provided. In this method, a compound is assayed for its ability to prevent or delay a temperature dependent phase transition in at least two distinct aqueous protein solutions. The aqueous protein solutions undergo phase transition to a condensed state upon either increasing or decreasing temperature of the solutions, and the method of assaying the aqueous protein solutions is preferably accomplished by measuring the temperature of condensation (T_c) for each of the aqueous protein solutions. Suitable proteins of the aqueous protein solutions include sickle hemoglobin and lysozyme.

In another aspect, a method for treating a phase transition disease state by administering an effective amount of a PCI is provided. Phase transition disease states include sickle cell anemia, Alzheimer's, cryoglobulinemia, rheumatoid arthritis and type II non-insulin dependent diabetes, and suitable PCIs of the present invention include those compounds identified by the above methods of identification.

Other aspects of this invention will become apparent upon reference to the attached figures and the following detailed description. Brief Description of the Drawings

Figure 1 illustrates that when both ΔE and ΔS_C are approximately constant with temperature, lowering the temperature below the temperature of condensation, T_c, favors protein condensation. Figures 2(A), 2(B), 2(C) and 2(D) are photomicrographs of a lysozyme solution in aqueous phosphate buffer at varying temperatures. Specifically, in Figure 2(A) the lysozyme solution is at a temperature of 6.6°C; in Figure 2(B) the temperature of the lysozyme solution has been lowered to -2.9°C; in Figure 2(C) the temperature of the lysozyme solution has been raised to a temperature of 1.6°C; and in Figure 2(D) the temperature of the lysozyme solution has further been raised to 36.1 °C.

Figure 3 illustrates the case when ΔE is dependent upon temperature and becomes increasing negative with increasing temperature, resulting in protein condensation upon raising the temperature of the protein solution.

Figure 4 illustrates the ability of a PCI of the present invention to lower T_c in the case of a protein which condenses upon lowering the temperature of the protein solution.

Figure 5 illustrates the ability of a PCI of the present invention to raise T_c in the case of a protein which condenses upon raising the temperature of the protein solution. Figure 6 illustrates the effect of a representative PCI of the present invention to inhibit the protein condensation of lysozyme.

Figure 7 is a photomicrograph illustrating the inhibition of sickle hemoglobin condensation by a representative PCI of this invention.

Detailed Description of the Invention

In brief, the present invention is generally directed to a protein condensation inhibitor ("PCI") which has utility in the treatment of disorders involving the phase transition of proteins from a normally soluble state to a condensed state, as well as to methods for identifying a compound for its ability to function as a PCI and to the use of such PCIs in the treatment of phase transition disease states.

As mentioned above, the present invention discloses a new paradigm for disease. A central distinguishing factor for a broad class of diseases involves a phase transition in which proteins, normally distributed in a single soluble phase, undergo a phase transition to a condensed state. As indicated above, the condensed state may be an amorphous or crystalline solid, a gel, a condensed liquid phase, a condensed liquid crystalline phase, or a distribution of protein clusters, aggregates or polymers of varying size. The transition of the protein into the condensed state plays a central role in the damage and loss of cellular and tissue function in disease states associated with a phase transition (referred to herein as "phase transition disease states"). Examples of such phase transition disease states include, but are not limited to, sickle cell anemia, Alzheimer's, cryoglobulinemia, rheumatoid arthritis, and type II non-insulin dependent diabetes.

The phase equilibria of cellular and extracellular proteins may generally be described using a phase diagram. The boundaries in such a phase diagram represent the conditions of temperature and concentration under which a protein solution undergoes a transition to the corresponding condensed state. In the practice of the present invention, the location of the protein condensation boundaries can be shifted by the introduction of an agent (referred to herein as a "protein condensation inhibitor" or "PCI"). A PCI of this invention interacts with the protein, or with the corresponding solvent molecules, such that under normal ambient physiologic conditions the protein remains in a soluble phase domain of its phase diagram. In other words, by changing the location of the phase boundaries by administration of a suitable PCI, the phase transition of the protein into a condensed state can be prevented.

In a related aspect, the kinetics of the protein phase transition may by modified in the practice of this invention. When a protein is placed under conditions such that a transition to a condensed state is thermodynamically possible, transport and collision processes will control the time constants required for establishment of the final equilibrium state. By the administration of a PCI of this invention, the length of time required for condensation of the protein may be sufficiently extended so as to maintain the protein in the soluble phase. In this manner, a PCI of this invention may function by slowing down the kinetics of the phase transition so as to substantially delay the actual condensation of the protein, even though the condensed state is thermodynamically preferred.

Accordingly, as used herein, a PCI of the present invention may function by either preventing protein condensation by shifting the protein phase diagram, or inhibiting protein condensation by impeding the kinetics thereof.

In general, proteins which undergo phase transition to a condensed state are abnormally altered as a result of post-translational or pre-translational (e.g., transcriptional or genetic mutation) modification of the protein's amino acid residues. Post-translational modification may be produced by chemical reactions after the protein is expressed and which alter the precise chemical structure of the protein residues on the surface or interior of the protein. Pre-translational modifications may be produced by errors in the sequence of the genes encoding the protein. For example, sickle cell anemia is caused by an error in the sequence of the hemoglobin gene. Instead of the normal hemoglobin protein (HbA), the gene produces the sickle hemoglobin (HbS) which differs from HbA by the substitution of a single amino acid (valine to glutamate) in each of the hemoglobin β chains. Upon deoxygenation, HbS undergoes a phase transition to form a condensed state ( a gel phase) containing long multi-stranded helical polymers (see, e.g., Eaton et al., Adv. Protein Chem. 40:63-279, 1990; Prouty et al.. J. Mol. Biol. 184:517-528.19851

A further example of a phase transition disease state is Alzheimer's. One of the proteins involved in this disease is β amyloid, a polypeptide of 42 amino acids. This protein is the product of the enzymatic cleavage of a 91 kilodalton protein, known as amyloid precursor protein. The β amyloid protein contains a strongly hydrophobic sequence which results in the formation of an insoluble amorphous fibrillar phase called amyloid plaque deposit. This deposit is associated with the presence of the clinical manifestation of Alzheimer's (see Halverson et al., Biochemistry 22:2639-2644, 1990). Type II non-insulin diabetes is another example of a phase transition disease state. In this disease, the islet amyloid polypeptide, amylin, produces amorphous solid-like deposits which are believed to be responsible for damage to islet β cells in the pancreas (Id). Other examples of phase transition disease states include cryoglobulinemia and rheumatoid arthritis. In the cases of cryoglobulinemia and rheumatoid arthritis, an important condensed state of protein is a complex or aggregation of immunoglobulins, particularly IgG and IgM (which act as an antigen and antibody, respectively). Complexes of these proteins (called rheumatoid factor) condense to form a precipitate at temperatures below 37°C (see Brandau et al., J. Biol. Chem. 261:16385-16389, 1986; Middaugh et al., Proc. Natl. Acad. Sci. USA 75:3440- 3444, 1978).

To appreciate the importance of the PCIs of the present invention, as well as the methods disclosed herein, a review of the thermodynamic basis of the phase transition of a protein to a condensed state is described below. In general, phase separation of aqueous, liquid protein solutions, and association or aggregation of proteins in such solutions are closely related phenomena in both physical nature and fundamental cause. They both include the formation of regions within the solution which have relatively high protein concentration, together with other regions having lower protein concentration. In the process of association or aggregation, the high concentration regions contain a relatively small number of initially separate protein molecules which stick together and move as a unit. These units can either remain suspended in solution, or comes out of the solution as a precipitate. In the process of phase separation, the high protein concentration phase can have a variety of physical forms, including liquid, amorphous or crystalline solid, and gelatinous. Each form is associated with a particular type of phase separation. For example, when both phases are liquid, the separation is termed "liquid-liquid phase separation." As used herein, the phase transition of a protein to a condensed state (also referred to herein as protein condensation) includes both protein aggregation and protein phase separation phenomena . The phase transition of a protein to a condensed state results from the net attraction of protein molecules for one another. Therefore, reagents which reduce this net attraction inhibit both protein aggregation and phase separation. The net attraction between proteins in solution may be expressed mathematically by the following equation (1):

The potential energy, ΔE, is a direct measure of the energetic advantage of having protein surrounded by protein and water surrounded by water, as compared to protein being surrounded by water molecules and vice versa. ΔE in general can be expected to be a negative number, and the more negative this negative number is, the greater the energetic advantage for condensation of protein and water into separate phases. In Equation (1), the magnitude of ΔE associated with the attraction of protein molecules is the sum of Epp, which denotes the potential energy of attraction between two protein molecules or portions thereof in close proximity, and E , which denotes the attraction between two water molecules, minus 2 times Ep ₅ which denotes the attraction between a protein molecule and a water molecule. Each such potential energy is taken relative to a reference state in which the relevant molecules are very far apart. Typically, each of the quantities E_PW, Epp and E will be negative in sign, representing attraction between each of the relevant pairs of molecules.

An equally important physical factor, quite apart from ΔE, opposes protein condensation. This factor arises because protein condensation greatly reduces the possible arrangements of the molecules in solution. This physical factor is expressed in terms of the absolute temperature, T, times the entropy reduction, ΔS_C, associated with protein condensation. TΔS_C is generally a negative number reflecting the joint roles of the temperature and the reduction in the number of configurations available to the protein and water molecules in the condensed state at temperature T. The fact that the quantity ΔS_C is a negative number reflects the fact that, upon condensation to form dense and dilute phases, the degree of disorder is reduced. Such a reduction is expressed in the reduction in entropy. The actual magnitude of ΔS_C depends on the precise nature of the condensation. Such condensation can include aggregation, liquid-liquid phase separation, gelation, liquid crystal formation, membrane formation and amorphous or crystalline solid formation, each of which will have its own appropriate value of ΔS_C.

The condition necessary for condensation can be expressed by comparing ΔE and TΔS_C. In general, if ΔE is a larger negative number than TΔS_C, then condensation can occur. If, on the other hand, ΔE is a smaller negative number than TΔ S_c, protein condensation of the given type does not occur. The mathematical expression of this consideration is set forth in equation (2):

Epp + E - 2E_PW < TΔS_C (2)

It should be noted that kinetic factors can control the transition to the thermodynamically favored state. That is, by controlling such kinetic factors it is possible to maintain the system is an uncondensed state even though the condensed phase is thermodynamically favored as expressed in equation (2).

The values of ΔE and ΔS_C may depend on solution conditions, including temperature and pressure, as well as the aqueous solvent, including pH and ionic strength. In view of the presence of the temperature component of equation (2), temperature T is an important factor in the phase transition of a protein to a condensed state. In the simplest case, ΔE and ΔS_C are approximately constant with temperature, and lowering the temperature reduces the absolute magnitude of the criterion value TΔS_C. Eventually TΔS_C will thereby be made less negative than Epp + E rw - 2 Epw, so that the condition in equation (2) is satisfied and condensation will be favored. This case is depicted in Figure 1, wherein the values of both ΔE and TΔS_C are plotted as functions of temperature. The intersection of the lines for ΔE and TΔS_C in Figure 1 corresponds to the condensation temperature, T_c, of the protein solution, which equals ΔE/ΔS_C. For temperatures below T_c, the dashed line of ΔE lies below the solid line of TΔS_C, and thus condensation will be favored. For temperatures less than T_c in this example, the energetic advantage of the condensation is greater than the entropic disadvantage of the condensation as reflected by ΔE being a larger negative number than TΔS_C, and as a result protein condensation is favored. On the other hand, for temperatures greater than T_c, T times the entropic disadvantage ΔS_C of condensation is so great that it overwhelms the energetic advantage in protein condensation. An example of condensation which occurs upon lowering temperature is illustrated by an aqueous solution of the protein lysozyme. This protein exhibits two condensation phenomena: crystallization and liquid-liquid phase separation. These phenomena are illustrated in Figures 2(A) through 2(D), wherein photomicrographs of a solution of lysozyme in aqueous phosphate buffer are presented. These figures show crystals of lysozyme, surrounded by an aqueous lysozyme solution. In Figure 2(A), the solution is held at a temperature below that for crystal formation, but above that for liquid-liquid phase separation (i.e., 6.6°C). Note the homogeneous regions in between the crystals reflecting the single phase nature of the solution contacting the crystals. When the temperature is then lowered (corresponding to moving to the left in Figure 1) the result is shown in Figure 2(B). In particular, upon lowering the temperature to a value of -2.9°C, which is below that of liquid-liquid phase separation, one can observe, in addition to the already condensed crystal phase, the roughening of the lysozyme solution corresponding to the separation of the solution into coexisting dense and dilute liquid phases. The reversibility of these two phase transitions is illustrated by raising the temperature (corresponding to moving to the right in Figure 1). In Figure 2(C), the temperature has been raised to 1.6°C so that now TΔS_C is actually less than ΔE. As a result, liquid-liquid condensation does not occur. This is seen in the photograph of Figure 2(C) by the clear, homogenous single phase characteristic of the solution in between the crystals. Finally, in Figure 2(D), the temperature has been raised sufficiently high (i.e., 36.1°C) so that it is above that corresponding to formation of lysozyme crystals. Under these conditions, the TΔS_C corresponding to formation of the solid protein phase is less than ΔE, and thermodynamic stability requires the melting of the crystals. It can be seen in Figure 2(D) that the crystals present in Figure 2(C) have started to dissolve. At this temperature, which is above that for the formation of crystals (at this overall concentration of lysozyme), the crystals will eventually dissolve completely. Other proteins which undergo condensation upon lowering the temperature include the cryoimmunoglobulin proteins which circulate in the bloodstream in the disease cryoglobulinemia, and which are often also found in rheumatoid arthritis.

If the quantity ΔE = E_PP + E_ww - 2 E_PW is dependent on temperature, protein condensation can occur upon raising the temperature, instead of upon lowering the temperature. This can occur when ΔE becomes increasingly negative with increasing temperature, so that it eventually becomes more negative than TΔS_C. This situation is illustrated in Figure 3, wherein the values of ΔE(T) and TΔS_C are plotted versus temperature. In this case, for temperatures below the temperature of condensation, T_c (i.e., the intersection of the graphs of ΔE and TΔS_C), the dashed line of ΔE lies above the solid line of TΔS_C, and thus no condensation of the ΔS_c-type will occur. For temperatures above T_c, the dashed line of ΔE lies below the solid line of TΔS_C, so that condensation will be favored. Sickle hemoglobin is an example of a protein which condenses upon raising the temperature.

As discussed above, the phase transition of a protein to a condensed state is an important aspect of the pathogenesis of the phase transition disease states of this invention, and which the PCIs of this invention prevent or inhibit. Equations (1) and (2) above, together with Figures 1 and 3, provide a context to identify the properties of a PCI according to this invention. For example, a PCI of this invention may bind to the protein in question, thereby changing the magnitudes of the quantities E_P and E_PP in Equation (1). Alternatively, the PCI may alter the properties of the aqueous solvent, thereby modifying E and Ep_W. In either case, the net result is an effective alteration in the magnitude of ΔE . Such changes correspond in Figures 1 and 3 to a vertical displacement of the dashed line representing ΔE .

The simplest case of such alteration by a PCI of this invention is one in which, at any temperature T, the magnitude of ΔE is changed by a constant amount, δE (i.e., ΔE'(T) = ΔE(T) + δE). Since larger negative values of ΔE favor protein condensation, when δE < 0 protein condensation will be enhanced, and where δE > 0 protein condensation will be inhibited. Thus, the PCIs of this invention, when administered to a patient suffering from a particular phase transition disease state, modify the protein and/or solvent associated with that disease state such that δE > 0. This principle is illustrated in Figures 4 and 5.

Specifically, Figure 4 illustrates the case wherein ΔE (T) is nearly constant as a function of temperature. As in Figure 1 , this case corresponds to the occurrence of protein condensation upon decreasing the temperature. Specifically, Figure 4 shows that the intersection point of ΔE + δE with TΔS_C has now moved from the original T_c to a lower temperature, (T_c)' < T_c. Thus, the PCIs of this invention lower T_c in the case of a protein which condenses upon decreasing the temperature. Similarly, Figure 5 illustrates the case of a protein which condenses upon increasing the temperature (which is the situation previously illustrated in Figure 3). In particular, Figure 5 shows that the new intersection point of ΔE + δE with TΔS_C, above which condensation will be thermodynamically favored, has moved from the original T_c to a higher temperature, (T_c)' > T_c. Thus, PCIs of this invention raise T_c in the case of a protein which condenses upon increasing temperature. In other words, a PCI which has the effect of increasing the value of ΔE to ΔE' = ΔE + δE, where δE >0 is relatively independent of temperature, will lower T_c for a lysozyme solution (which condenses when the temperature is lowered), but will raise T_c for a sickle hemoglobin solution (which condenses when the temperature is raised). In either case, the PCI will prevent or inhibit the phase transition of the respective proteins to their corresponding condensed states.

Accordingly, an essential feature of a PCI of this invention may be represented by a shift in the value of ΔE to a new value which is above the original value. That is, the new value ΔE + δE is not as large a negative number as is ΔE. Thus, a PCI of the present invention reduces the numerical magnitude of the quantity ΔE, which represents the driving force associated with protein condensation. This corresponds to a positive value of the quantity δE - that is, the value δE > 0.

In the above disclosure, two possible temperature dependencies for ΔE have been considered, and it has been assumed that ΔS_C was independent of temperature. However, such circumstances may be considered as representative examples of the more general situation in which both ΔE and ΔS_C can be strongly dependent on the temperature. Regardless of the particular temperature dependence of ΔE or ΔS_C, the general condition that ΔF_C = ΔE(T) - TΔS_C(T) < 0, where ΔF_C denotes the free energy of condensation, is the condition for the formation of a condensed phase to be thermodynamically favored. Using the quantity ΔF_C, one may determine the change T_c' - T_c in phase separation temperature resulting from a reduction in the magnitude of the quantity ΔE, for any assumed form of the temperature dependence of ΔS_C(T) and ΔE(T). If the change in ΔE is δE, then the corresponding change in T_c is given by equation (3):

(T_c' - T) = - δE/(dΔE/dT - d(TΔS_c)/dT) (3)

In equation (3), the derivatives are evaluated at the original temperature of condensation, T_c. The denominator in this expression is precisely (dΔF_c/dT) evaluated at T_c. Thus, since the criteria for protein condensation to be thermodynamically favored is that ΔF_C < 0, this denominator will be positive when condensation occurs upon lowering the temperature. The sign of the denominator may

^' be understood by realizing that ΔF_C would increase to a value > 0 if the temperature were raised above T_c. In contrast, (dΔF_c/dT) will be negative when condensation occurs upon raising the temperature. For a substance to be a PCI in the context of this invention, the value of δE must be positive. Thus, equation (3) provides a basis for experimental identification of PCIs since the quantity T_c' - T_c can be measured, and since the sign of the denominator can be determined from experimental measurement of whether condensation occurs upon raising or lowering the temperature, as described above. That is, one can experimentally determine for any given compound whether it is a PCI for a particular protein solution by determining the sign of δE as deduced using equation (3). Turning to certain embodiments of this invention, a method is disclosed for identifying a compound which functions as a PCI. In one aspect of this invention, the method includes assaying the compound in an aqueous protein solution to determine whether the sign of δE, as discussed above, is positive. Such assaying may include the steps of measuring a change in phase separation temperature of the aqueous protein solution, T_c'-T_c, by the addition of the compound to the solution, and also determining whether protein condensation of the aqueous protein solution occurs upon raising or lowering its temperature. In short, equation (3) above is employed for experimental identification of PCIs. In a preferred embodiment of this method, the aqueous protein solution contains (preferably exclusively) a protein associated with a phase transition disease state. Such proteins include, but are not limited to, sickle hemoglobin which is associated with sickle cell anemia.

In another aspect of this invention, a method is disclosed for identifying a compound which functions as a PCI by individually assaying the compound for its ability to prevent or delay a temperature dependent phase transition in at least two distinct aqueous protein solutions which undergo a phase transition to a condensed state upon raising or lowering the solution temperature. Compounds that function as PCIs may thus be identified using two or more protein solutions. This permits the use of readily available proteins to identify compounds which function as PCIs. Moreover, this method can be used even with proteins whose condensation are not associated with disease states. The ability of a compound to function as a PCI in two distinct protein solutions indicates its utility for preventing or inhibiting condensation of proteins associated with phase transition disease states. The protein used for one solution must be a different protein from that used for the other solution (or solutions). An example of two such distinct proteins is lysozyme (which condenses upon decreasing the temperature) and sickle hemoglobin (which condenses upon increasing the temperature). Preferably, the compound is tested for its effect on the temperature of condensation, T_c, in both of the protein solutions, and the compound is a PCI if its effect on both protein solutions corresponds to δE > 0. (Alternative methods for testing the effectiveness of a compound as a PCI, rather than direct measurement of Tc, are discussed in greater detail below.) At any given temperature, and particularly at body temperature, the effect of a PCI of this invention is to increase the value of the quantity ΔE - TΔS_C to the new value ΔE + δE - TΔS_C. Compounds satisfying the above test will inhibit the thermodynamic factors which favor the condensation of proteins associated with phase transition disease states. The effect of a compound on T_c of a protein solution can be most readily determined when protein condensation occurs without significant delay following a change in temperature. In the case of liquid-liquid phase separation, in which the condensed phase is a concentrated liquid protein solution, while the dilute phase is a less concentrated liquid protein solution, the difference in index of refraction between the phases leads to intense scattering of light. This scattering can be detected as an increase in the intensity of light scattered by the protein solution. Alternatively, it can be detected as a decrease in the intensity of light transmitted through the solution. The temperature dependence of these scattered or transmitted light intensities can be used to defined the temperature of condensation, T_c. As explained above, if protein condensation occurs upon lowering the protein solution temperature, a PCI will lower T_c, while if protein condensation occurs upon raising the protein solution temperature, the PCI will raise T_c.

Alternatively, even if one cannot directly operate experimentally at the phase separation temperature, one can determine its value by noting that the intensity of the scattered light will, in general, increase as one approaches the phase boundary. In fact, the intensity of the scattered light can be used to measure the quantity ΔF_C used above. For example, in the case of liquid-liquid phase separation, the intensity of the scattered light is inversely proportional to the appropriate ΔF_C, when ΔF_C is > 0. Thus, measurements of the intensity of the scattered light as a function of temperature for a protein solution, reflecting the dependence of ΔF_C upon temperature, permit determination of an effective value of T_c corresponding to a temperature where ΔF_C would be zero, and likewise determination of the change in δE upon adding the compound of interest to a given protein solution. Other methods of determining whether a compound is a PCI of this invention may also be utilized. For example, as simple aggregation of proteins proceeds, there will generally be a great increase in the amount of light scattered by the solution, especially as the size of the aggregates approaches the wavelength of light. By measuring such light scattering, compounds may be evaluated for their effectiveness as a PCI. Moreover, the temperature of the appearance of the condensed phase can be directly monitored by temperature-controlled microscopy (as illustrated in Figure 2), and the effectiveness of a compound as a PCI can be monitored by analysis of the resulting images. Alternatively, thermodynamic measurements of ΔF_C can be made. These include measurement of the osmotic pressure π of the solution, which permits the reconstruction of the free energy, and/or measurements of the vapor pressure of water in equilibrium with the solution. Calorimetry can also be used to directly measure the heat given off by the solution upon condensation. NMR, electron microscopy or X-ray scattering can also be used to monitor the appearance of the condensed phase. Furthermore, mechanical properties of the solution can be measured which are sensitive to the condensation. Some protein condensation, as in the case with sickle hemoglobin, involves the formation of a gel having pronounced viscoelasticity and resistance to flow. The measurement of the above properties can be accomplished using known techniques, including the procedures disclosed in the following references: Cantor and Schimmell, Biophysical Chemistry - Techniques for the Study of Biological Structure and Function, part 2, Freeman publisher, N.Y., 1980; Hiemenz, Principles of Colloid and Surface Chemistry. 2nd ed., Marcel Dekker publisher, N.Y., 1986; and Ke vonHolde, Physical Biochemistry, 2nd edition, Prentice-Hall publisher, Englewood Cliffs, N.J., 1985 (which references are incorporated herein by reference in their entirety).

A representative example of a PCI of this invention is pantethine. As illustrated in Example 1 herein below, the ability of pantethine to inhibit protein condensation in aqueous solutions of lysozyme is demonstrated. In such solutions pantethine lowered T_c by approximately 105°C/Molar, as illustrated in Figure 6 by a plot of the change in T_c (vertically axis) against the concentration of pantethine (horizontally axis). In addition, pantethine was also tested on solutions of the protein, sickle hemoglobin, as illustrated in Example 2 below. In this example, the temperature of condensation of solutions of the sickle hemoglobin was raised by an amount which exceeded 241°C/Molar. Thus, the effect of pantethine on both of these proteins is consistent with δE > 0. Moreover, the two proteins tested were quite different from one another, as demonstrated by the fact that one condenses upon lowering, and the other upon raising, the temperature of the solution. From these results, PCIs of this invention, including pantethine, inhibit the condensation of the specific proteins associated with phase transition disease states.

The PCIs of this invention, including pantethine, may be administered to a patient by any manner of accepted modes of administration, including (but not limited to) parenterally (e.g., by injection, topical administration and inhalation) and orally. In the practice of this invention, an effective amount of a PCI is administered to the patient having a phase transition disease state. An effective amount of PCI is any amount sufficient to prevent or delay the phase transition to a condensed state of the protein associated with a given phase transition disease state. The specific formulation of the PCI will depend upon the intended mode of administration. For example, PCIs may be formulated as a solid, semi-solid or liquid, such as, for example, tablets, pills, capsules, powders, or suspensions. In addition, the formulation may include one or more pharmaceutically acceptable carriers and/or diluents. Formulations intended for parenteral administration may contain wetting or emulsifying agents and pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, or triethanolamine oleate. Suitable carriers for liquid compositions include, for example, water, saline, glycerol, ethanol, and aqueous dextrose in an amount sufficient to form a solution or suspension. For solid compositions, suitable carriers include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate. Other carriers, and methods for preparing the dosage forms will be apparent to those skilled in the art.

The following examples are offered by way of illustration, not limitation.

EXAMPLES Example 1

Effect of PCI on Lysozyme Protein Solution

Lysozyme was obtained from Sigma Chemical Company and dialyzed into 0.1 M sodium phosphate buffer (pH 7). The lysozyme solution was then concentrated by ultra filtration to stock concentrations which show phase separation, and stored at 4°C until use. Pantethine was added to the lysozyme solution to yield final pantethine concentrations of OmM, lOmM, 15mM and 25mM. These solutions were held at a temperature above the condensation temperature, and the light transmitted through the solution was measured while lowering the temperature. The temperature at which the initial transmitted light dropped to 50% its original value was used as the temperature of condensation, T_c. Figure 6 illustrates the change in T_c as a function of pantethine concentration. A linear fit of the change in T_c as a function of pantethine concentration yields a slope of -105°C/Molar, illustrating that pantethine is a PCI for lysozyme solutions.

Example 2 Effect of PCI on Sickle Hemoglobin Protein Solution

A. Phase Transition of Deoxygenated HbS to a Condensed State HbS was obtained from Sigma Chemical Company and dialyzed into

0.15 M potassium phosphate buffer (pH 7.14), for two days to remove salts and other impurities. The HbS solution was then concentrated by ultra filtration to stock concentrations above 200 mg/ml, and stored at 4°C until use. The concentration of HbS in the stock solutions was determined by visible wavelength absorption spectroscopy, after conversion of the HbS to the cyanmethemoglobin form, according to the method of Van Assendelft (Spectrophotometry of Hemoglobin Derivatives, Koninklijke Van Gorcum, The Netherlands, 1970) (incorporated herein by reference). Prior to deoxygenation with sodium dithionite, the gas above each solution was purged of oxygen using a humidified stream of nitrogen, applied to each sample with needles, through a rubber septum. Sodium dithionite (Na2S2U4) was obtained from Sigma Chemical

Company and stored in a nitrogen filled desiccator. Solutions were prepared fresh daily, under nitrogen, from the stock dithionite. Deoxygenation of HbS solutions was carried out on an ice bath, in a nitrogen filled glove bag to prevent oxygenation of the samples or exposure of the dithionite solutions to oxygen. Carrying out the deoxygenation at a low temperature prevented gelation from occurring before the sealed samples were removed from the glove box. To deoxygenate the HbS solutions, Na2S2θ4 was added to obtain a final concentration of 0.03 to 0.04 M, providing approximately for a 3:1 molar ratio of Na2S2θ4:heme group (there are four heme groups per HbS molecule). In one experiment, a solution of 207 mg/ml HbS was deoxygenated, and then warmed to room temperature (22°-23°C). The HbS solution gelled quickly at room temperature, and remained almost entirely gelled after incubating over night at 4° C. In a second experiment, a solution of 195 mg/ml HbS gelled after 2-3 minutes at 40° C. This gelation was reversed at 4°C. The sample was subsequently placed at room temperature, and gelation occurred after 3 hours, 50 minutes. These results demonstrate that in vitro deoxygenation of HbS with a2S2θ4 results in gelation at higher temperatures, which is reversible upon cooling.

B. Inhibition of HbS Phase Transition by Pantethine HbS solutions at concentrations of 194-197 mg/ml were prepared as described in Section A above. Prior to deoxygenation, pantethine was added to four of the samples to yield concentrations of 32 mM, 58 mM, 101 mM and 154 mM. No pantethine was added to the fifth sample to serve as a control. After deoxygenation, all samples remained liquid at 4°C, after heating in hands (i.e., gripping a test tube containing the sample in the palm of the hand) for 5 minutes, and after heating in a water bath at approximately 45°C for 5 minutes. However, when the samples were subsequently placed at 22-23 °C for 20 minutes, the sample without pantethine gelled, while the other solutions remained liquid. The sample containing 32 mM pantethine gelled slowly at 37°C, but remained liquid for more than 18 hours at 30°C. The samples containing 58 mM, 101 mM and 154 mM of pantethine did not gel at or below 37°C. The results of this experiment are summarized in Table 1, and illustrate that pantethine inhibits the deoxygenation-induced gelation of HbS. In Table 1, the conditions for temperature and time of incubation were employed successively.

Table 1 Temperature and Time Pantethine concentrations of incubation 0 mM 3 mM 101 mM 154 ml

4°C, 20 min. liq. liq liq liq. hands, 5 min. liq. liq liq liq.

45°C, 3 min. liq. liq liq liq. 22-23°C, 20 min. gel liq liq liq.

22-23°C, 50 min. gel liq liq liq.

37°C, 90 min. gel liq liq liq.

23°C, 2 min. gel liq liq liq.

37°C, 60 min. gel liq liq liq. 37°C, 90 min. gel liq liq liq.

23 °C, 2 min. gel liq liq liq.

4°C, 30 min. liq. liq liq liq.

30°C, 90 min. liq. liq liq liq.

30°C, 17 hrs. gel liq liq liq. 4°C, 1 week liq. liq liq liq. 22-23 °C, 10-20 min. gel liq liq liq.

To further demonstrate the ability of pantethine to prevent or delay the phase transition of HbS to the condensed state, two capillary tubes were each filled with a solution containing 195 mg/ml of HbS in an 0.15M aqueous potassium phosphate buffer. One tube also contained 203mM of pantethine. The HbS solutions contained in both tubes had previously been deoxygenated using sodium dithionite as described above in Section A. The tubes were then placed on a microscope stage and brought to a temperature of 37.4°C, and a photograph of the tubes is presented in Figure 7. In this figure, the tube on the left contained pantethine. As illustrated in Figure 7, pronounced concentration fluctuations are evident from the uneven brightness within the right hand tube at this temperature, which does not contain pantethine. These fluctuations correspond to the formation of a mixture of condensed phase of the protein having high refractive index, together with a dilute phase having low refractive index. Comparable fluctuations are not seen in the tube on the left, which contains pantethine, illustrating the inhibition of protein condensation by pantethine and, thus, that pantethine is a PCI for sickle hemoglobin solutions.

From the foregoing, it will be appreciated that, although specific embodiments of this invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

Claims

1. A method for identifying a compound which functions as a protein condensation inhibitor, comprising assaying a compound in an aqueous protein solution to determine whether the sign of δE is positive.

2. The method of claim 1 wherein the assaying includes the steps of measuring a change in phase separation temperature and determining whether protein condensation occurs upon raising or lowering the temperature of the aqueous protein solution.

3. The method of claim 1 wherein the aqueous protein solution comprises a protein associated with a phase transition disease state.

4. The method of claim 3 wherein the protein is sickle hemoglobin.

5. A method for identifying a compound which functions as a protein condensation inhibitor, comprising individually assaying a compound for its ability to prevent or delay a temperature dependent phase transition in at least two distinct aqueous protein solutions.

6. The method of claim 5 wherein at least one of the distinct aqueous protein solutions undergoes a phase transition to a condensed state upon increasing the temperature of the solution.

7. The method of claim 5 wherein at least one of the distinct aqueous protein solutions undergoes a phase transition to a condensed state upon decreasing the temperature of the solution.

8. The method of claim 6 wherein the aqueous protein solution which undergoes a phase transition to the condensed state upon increasing temperature is a sickle hemoglobin solution.

9. The method of claim 7 wherein the aqueous protein solution which undergoes a phase transition to the condensed state upon decreasing temperature is a lysozyme solution.

10. The method of claim 5 wherein the individual assaying of the compound is accomplished by measuring the effect of the compound on the temperature of condensation of the distinct aqueous protein solutions.

11. The method of claim 10 wherein the effect of the compound on the temperature of condensation is determined by measurement of the distinct aqueous solutions using techniques which measure light transmittance, light scattering, osmotic pressure, water vapor pressure, heat of condensation, NMR, X-ray scattering, electron microscopy, light microscopy, viscoelasticity, resistance to flow and gel formation.

12. The method of claim 10 wherein the effect of the compound on the temperature of condensation is determined by measuring the change in the light transmittance through the distinct aqueous protein solutions.

13. The method of claim 10 wherein the effect of the compound on the temperature of condensation is determined by measuring the change in the light scattered by the distinct aqueous protein solutions.

14. A method for treating a phase transition disease state, comprising administering to a patient an effective amount of a protein condensation inhibitor identified by claim 1 or claim 5.

15. The method of claim 14 wherein the phase transition disease state is selected from sickle cell anemia, Alzheimer's, cryoglobulinemia, rheumatoid arthritis and type II non-insulin dependent diabetes.

16. The method of claim 14 wherein the phase transition disease state is sickle cell anemia.

17. The method of claim 14 wherein the phase transition disease state is Alzheimer's disease.

18. The method of claim 14 wherein the phase transition disease state is cryoglobulinemia.

19. The method of claim 14 wherein the phase transition disease state is rheumatoid arthritis.

20. The method of claim 14 wherein the phase transition disease state is type II non-insulin dependent diabetes.

21. The method of claim 14 wherein the protein condensation inhibitor is pantethine.