Patent 6,495,338

The present invention relates to a method for screening drugs for use in treating hypertension using the tubular renin-angiotensinogen system identified by the present invention. The invention further relates to a method to diagnose sodium status in an individual by measuring urinary angiotensinogen, angiotensin-I, des-AI-angiotensinogen or renin.

Definition of Sodium Sensitivity

Epidemiology, physiology, pathology and drug response indicate that essential hypertension encompasses a variety of conditions of unknown cause that cannot be resolved on clinical grounds alone. An important physiological distinction is whether or not sodium salt plays a significant contribution to disease. A dominant hypothesis is that there are innate differences in individual response to excess dietary sodium, and this factor would account for a significant proportion of cases of essential hypertension. This class can be defined a "sodium sensitive" hypertension simply to stress the role of this contributing factor in the development of high blood pressure.

While the significance of sodium in the epidemiology of essential hypertension is compelling in the aggregate, the relationship between sodium consumption and blood pressure has been difficult to establish at the individual level. A variety of protocols have been designed in an attempt to identify individuals who would be particularly vulnerable to excess sodium consumption. Typical maneuvers include blood pressure or weight response to a standardized sodium load. Another approach has been to monitor change in renal blood flow after infusion of angiotensin-II in individuals exposed to a high sodium diet, or similar physiological manipulations. In general, these approaches have confirmed that there are indeed two broad classes of responses to such maneuvers, some response or none, leading to the definition of "sodium sensitive" and "sodium resistant" individuals. The overlap between the two groups remains so large, however, that it precludes the unambiguous identification of any particular individual as a member of either group. This diagnosis issue has been particularly vexing for medical practice, as the efficacy of either dietary sodium restriction or specific therapeutic intervention critically depends on the identification of this underlying factor.

Sodium sensitivity, then, measures an individual's propensity to respond to excess sodium intake by an increase in either blood pressure or weight. In the context of a chronic condition such ss essential hypertension, which develops insidiously over decades, and where the adverse consequences of excess sodium intake are reflecting minimal but cumulative attrition over time, it is not altogether surprising that such a differential in chronic response escapes characterization by an acute maneuver. Related concepts will be defined below.

Sodium homeostasis subsumes the overall mechanism by which the body regulates the fate of sodium as a function of physiological needs. Sodium balance represents the net difference between intake and excretion which, on average, is zero. In certain situations, such as pregnancy or after significant blood loss, intake exceeds excretion to accommodate volume expansion or reexpansion. Sodium status, although almost synonymous with sodium balance, is generally used to characterize dietary sodium status, namely sodium excess, normal sodium, or restricted sodium intake. Monitoring sodium status is important before performing clinical maneuvers as described above, or more relevant yet to monitor compliance to dietary sodium restriction. If genetic differences contribute to sodium sensitivity, then it would be clearly of clinical relevance to characterize sodium sensitivity as a genetic "liability," or an innate predisposition to develop high blood pressure.

Significance of Intrarenal A-II in Regulation of Sodium Balance

The link inferred between angiotensinogen and sodium homeostasis results from its known physiological function. Angiotensin-II (A-II), generated exclusively from angiotensinogen protein (ANG) through two steps of enzymatic cleavages catalyzed by renin and angiotensin-converting enzyme (ACE), exerts short-term and long-term effects on vascular tone and blood volume and, as a result, it is a major determinant of blood pressure. As we argue here, the short-term effects of A-II are better understood than are its long-term effects.

The former reflects the vasoconstrictor effect of A-II at the systemic level. Specifically, renin made by a specialized segment of afferent renal arteries (called the juxtaglomerular apparatus, or JGA) acts in the general circulation on angiotensinogen released by the liver to form angiotensin-I (A-I), subsequently converted to A-II by ACE in capillary vessels. Increased circulating A-II would then induce constriction of arterioles that regulate peripheral vascular resistance and the overall compliance of the vascular system. When blood volume is depleted, the net effect of reduced compliance is maintenance of normal blood pressure. When blood volume is normal, increased vascular resistance leads to increased arterial pressure.

The characterization of the long-term effects of A-II has proven far more elusive. Reexpansion of blood volume after depletion requires sodium retention in the kidney (as water "follows" sodium). Under normal conditions, variation in dietary sodium intake leads to compensatory adjustment of sodium excretion so as to maintain baseline blood pressure within narrow limits. A-II has been recognized as the dominant hormone promoting sodium retention, through both indirect and direct renal effects.

Indirect effects of A-II are mediated by aldosterone, a mineralocorticoid (a steroid affecting mineral metabolism) released by the adrenal after A-II stimulation. This hormone acts in the distal part of the nephron where it promotes sodium reabsorption and potassium excretion. Most textbooks still emphasize the presumed dominance of aldosterone in promoting sodium retention.

The direct sodium-retaining effects of A-II in the kidney are multiple and varied, affecting both renal hemodynamics, that is, the regulation of blood flow through various parts of the kidney, and the activity of sodium transporters mediating reabsorption of filtered sodium. These direct effects have been primarily demonstrated by addition of A-II to experimental preparations, and as such, these experiments have not clarified the actual origin, site of action, and regulation of A-II accounting for such effects.

A large body of experimental evidence accumulated over the last two decades has demonstrated that, in normal physiological states, aldosterone plays only a modest role in regulation of sodium excretion to balance intake. Rather, this function is primarily mediated by intrarenal A-II.

A Paracrine Tubular Renin-Angiotensin System

The essence of the findings described here is as follows:

(1) Angiotensinogen is secreted into tubular fluid by epithelial cells of the proximal tubule (cells lining the luminal side of this nephron segment).

(2) AGT expression at this site is a function of sodium status.

(3) Angiotensinogen protein transits through the entire nephron and can be measured in urine, where it results from proximal tubule secretion (circulating angiotensinogen is not filtered through the glomerular membrane, by contrast to renin).

(4) Renin is expressed by principal cells of the distal nephron, the very cells expressing both sodium channel (for sodium reabsorption) an potassium channel (for potassium secretion under control of aldosterone).

(5) Renin expression at this site also varies as a function of sodium status.

(6) Blocking the sodium channel of principal cells with amiloride leads to up-regulation of renin expression in distal nephron, indicating that sodium translocated by this channel is an important sensing mechanism in the regulation of renin expression in distal nephron.

(7) A-I and active renin, reflecting activity of distal nephron, can be measured in urine.

Proposed Function of This Paracrine System

Massive amounts of sodium are filtered daily, of which 99%, to almost 100% are reabsorbed by the kidney ("Sodium Balance"). Different transporters are involved in various segments of the nephron ("Sodium Transporters"), and coordination of the activity in each segment determines the final amount of sodium excreted in final urine. It is common to contrast the functions of proximal and distal tubule as "bulk" and "fine" sodium reabsorption, respectively. After a bulk phase where the majority of filtered sodium, water and various solutes are reabsorbed together by a global process dominated by sodium movement, a fine phase allows independent, fine adjustment of each constituent in final urine. Angiotensinogen expression in proximal tubule suggested its involvement in the bulk phase, but did not provide a mechanism for a more critical role in final adjustment of urinary sodium.

The paracrine system described herein provides an answer. It reveals the mechanism by which the renin-angiotensin system regulates sodium excretion by integrating function at these two critical sites. The concept is novel, and it has extensive implications for diagnosis therapeutic research. The diagnostic implications will be detailed, rather briefly given previous claims and the background given here.

As shown by the examples below, it has been found that proximal tubular epithelium cells synthesize and secrete angiotensinogen, that angiotensinogen circulates through the entire nephron and can be detected in urine, that renin is expressed by principal cells of the distal nephron, and that expression of substrate and enzyme at these sites is affected by variation in dietary sodium. This previously unidentified tubular renin-angiotensin system provides the basis for the drug screening method of the present invention.

A large number of pharmaceutical drugs have been developed and are used as antihypertensive agents. They can be classified into broad subclasses as a function of their principle of action and the biochemical function they target. As noted above, the renin-angiotensin system (RAS) is of fundamental importance in blood pressure control. The most recent drugs developed in the field interfere with this system in one of at least three ways. Renin inhibitors are analogs of the angiotensinogen cleavage site which bind to renin with high affinity, and as such, compete with angiotensinogen. Although effective, these compounds have been of limited usefulness because of problems in drug delivery. ACE inhibitors, such as captopril or lisinopril, have proven effective and have become one of the drugs of choice in the treatment of essential hypertension. The A-II Type 1 receptor inhibitor, Losartan from Dupont-Merck, represents the newest agent developed to counter the physiological effects of A-II. Its high affinity for the major receptor mediating the hemodynamic effects of A-II accounts for its action.

A common feature of these drugs designed to interfere with the RAS is that they have global, systemic and local effects. Indeed, given the preeminent role ascribed to the circulating RAS in research preceding the development of these agents, they reflect the state of knowledge of the time at which drug development was developed in these directions.

The results described herein pertaining to the existence and the role of a paracrine tubular RAS in the regulation of sodium balance suggest new targets for therapeutic intervention and new methods to screen compounds and ascertain their biological effectiveness.

Thus, the present invention identifies novel targets for the development of antihypertensive agents. The new agents will primarily interfere with the normal function of the paracrine tubular RAS we describe at either the proximal tubule or in the distal nephron. Compounds can be engineered so as to be delivered at either site and so that their biological activity is optimal under the prevailing environment of each segment. The net effect is to control sodium reabsorption, and as such, it will prevent the development of essential hypertension in subjects deemed sodium sensitive. The drugs may prove most effective in a subset of hypertensive patients. Together with means to identify such subjects, as we have claimed with AGT the selectivity and specificity of such drugs will alleviate the difficulty of choosing a given drug and determining effective dosage. It is recognized that any given drug is effective in only a fraction of patients, and at present, there is no simple way of predicting if any given patient will respond well to any particular agent. Indications may be based on associated manifestations, such as coronary heart disease, but not on actual knowledge about the mechanism accounting for hypertension. Both conditions being common, there must be instances where hypertension depends on factors distinct from those accounting for coronary disease.

In addition, screening methods are used to determine the efficacy of compounds designed so as to interfere with the renin-angiotensin system at either the proximal or distal tubule. The effects of these compounds can be monitored at three levels: cellular, tissue and whole organism. In the proximal tubule, cellular response to drugs can be monitored in terms of angiotensinogen expression and secretion or in terms of sodium transport by the sodium-hydrogen exchanger and other sodium-dependent transporters. In the distal tubule, targeted drugs will affect the activity of the sodium channel, the density of A-I receptors, and the synthesis and release of renin by principal cells. At the tissue level, expression of angiotensinogen and renin can be monitored by any one of the methods described herein, including in situ RT-PCR, RT-PCR of microdissected nephron segments, particularly Y-junctions, and immunohistochemistry. At the level of the entire organism, the efficacy of compounds can be evaluated by measuring parameters of the paracrine RAS in urine, including A-I and A-II total angiotensinogen, des-AI-angiotensinogen, uncleaved angiotensinogen, total renin and renin activity. Furthermore, the effects of such agents on blood pressure and plasma volume can be monitored.

As shown by the examples below, it has been found that angiotensinogen, its enzyme catalyzed products or renin excreted in urine vary with changes in dietary sodium. Thus, the sodium status of an individual is diagnosed by determining the amount of angiotensinogen or its enzyme catalyzed products or renin in the urine of the individual and comparing the determined amount with normal values. Any method for detecting urinary angiotensinogen or angiotensin-I can be used in accordance with the present invention. A finding of elevated levels of these compounds indicates high sodium. The levels of these compounds are determined using conventional techniques and any appropriate method is suitable for use. An individual's sodium sensitivity can also be determined by determining the amounts of these compounds in urine. If the levels are elevated in an individual under a high salt diet compared to reference value, the individual is sensitive to salt.

It has also been found that expression of angiotensinogen and renin is regulated at specific sites along the kidney. This finding identifies new therapeutic targets for blood pressure control and provides a basis for a method to screen drugs for use in treating hypertension. According to the present invention, drug candidates for treating hypertension are screened using the tubular renin-angiotensin system by testing the effects of drug candidates at the proximal and/or distal tubule.

Molecular variants in the angiotensinogen gene (AGT) may reflect individual predisposition to the development of essential hypertension, as we claimed earlier with t235 and a(-6) variants. The actual manifestation of the genetic propensity evidently depends on the degree and the duration of the exposure to high sodium intake as well as other promoting factors such as overweight and excess stress.

Angiotensinogen, A-I and active renin can be measured in urine in animals and humans. Furthermore, the amount of angiotensinogen detected in urine reflected sodium status. It was at the limits of detection under high sodium diet, but high under sodium restriction. Measuring angiotensinogen and related parameters in the urine should provide clinical indicators of the activity of this paracrine tubular RAS. Not only should these correlate with sodium status, but they may also serve as markers of sodium sensitivity. Indeed, the hypothesis derived from work on A(-6) AGT mutation is that individuals homozygous for this variant would tend to maintain greater AGT expression under a high sodium diet than would individuals of other genotypes. This modest differential in the ability to down-regulate AGT under excess sodium would account for a relative propensity to retain more sodium, with long-term attendant effects on blood pressure. This would account for sodium sensitivity in these individuals. Not only could A(-6) genotype serve as a marker of this liability, as claimed earlier, but also, it is more likely that urinary parameters reflecting the activity of this newly identified paracrine tubular RAS may prove of clinical value to identify sodium sensitive individuals. These individuals would stand a higher risk of developing essential hypertension when confronted with the high sodium diet characteristic of affluent societies.

Certain parameters of the paracrine RAS can be measured in urine, specifically A-I, ANG, des-AI-ANG, and active renin (ANG denotes uncleaved, entire angiotensinogen protein, des-AI-ANG is the complement of the ANG protein after AI has been cleaved; as the peptide AI is expected to be less stable and to degrade rapidly, ANG + des-AI-ANG reflect the total amount of ANG produced in proximal tubule). These parameters reflect an individual's sodium status, as ANG and renin are down-regulated or up-regulated under high or low sodium, respectively. An individual can be classified as sodium sensitive if levels of AI, ANG, or des-AI-ANG are elevated under high salt diet compared to a reference series of individuals. The parameters names above may correlate to genetic predisposition to essential hypertension measured by AGT genotypes (M/T235 or A/G(-6)). These parameters may also be of diagnostic value in a number of clinical instances, including minimal renal disease, diabetic nephropathy, IgA nephropathy, and disorders likely to affect the function of the paracrine tubular RAS described in this application.

Claim 1 of 2 CLaims

What is claimed is:

1. A method for determining the sensitivity of an individual to sodium, wherein sodium sensitivity is defined as an individual's tendency to respond to excess sodium intake with an increase in blood pressure, said method comprises measuring the amount of a substance selected from the group consisting of angiotensinogen, angiotensin-I and des-angiotensin-I-angiotensinogen in the urine of an individual under normal physiological conditions except said individual is on a high salt diet and comparing said amount with a reference standard, wherein an elevated amount of said substance in an individual on a high salt diet under normal physiological conditions is indicative of sodium sensitivity.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]