CA2192855A1 - Improved methods for the determination and adjustment of prolactin daily rhythms - Google Patents

Abstract

Disclosed are methods for detecting and adjusting abnormalities in prolactin daily rhythms. Provided is a method comparing a prolactin profile of a vertebrate subject to the prolactin profile of other vertebrate subjects having a normal prolactin profile; and adjusting the prolactin profile of the subject to cause the profile of the subject to conform to (or approach) the normal prolactin profile. Also provided is a method for evaluating the daily prolactin profile of a human subject wherein the method provides for comparing a set of at least two prolactin levels of the human and comparing them to corresponding prolactin levels for healthy humans (or a preestablished average prolactin curve for healthy humans) at the same time points. The method also provides for determining whether a human subject has an abnormal prolactin profile.

Description

W096/00396 r~
~1 2 1 92~55 IMPROVED METHODS EOR T~E D~ NATION
AND ADuu~ OF PROLA~TIN 3AI~Y REY~Mc FIELD OF THE ~Nv~ ~N
This invention relates to improved methods for the reduction in a subject, vertebrate animal or human, of body fat stores, and r-eduction of at least one of insulin resistance~ hyperinslll;n~iA, hyperlipidemia, hyperglyce-mia, and other metabolic diseases, especially those associ-ated with Type II diabetes. In particular the present invention is directed to methods for: (i) normalizing the daily prolactin rhythms of a human or vertebrate animal;
(ii) diagnosing aberrant daily prolactin rhythms of a human or vertebrate animal; and (iii) determining the appropriate adjustments that need to be made to normalize such aberrant prolactin rhythms. Such adjustmgnts include the daily administration to the subject of at least one of a prolac-tin stimulator-and/or a prolactin inhibitor at a predeter-mlned time of day (if only one is administered) or atdifferent predetermined times of day (if both are adminis-tered~. This therapy typically results in the long-term adjustment of~aberrant or abnormal prolactin rhythms so that they ccnform to or simulate normal prolactin cycles, at which point the therapy may be discontinued, while the adjustment pcrmits.
This invention is also directed to improved ~ methods for adjusting the neural oscillator (or oscilla-tors) of which the daily prolactin rhythm is an expre-ssion ~ ~o or marker.
The adjustment of daily prolactin rhythms and the daily rhythms of key neural oscillators results in reduc-W096/00396 2 1 9 2 8 5 5 P~ 3~-1 tion and control over an extended time period of various metabolic or other disorders.

BACRGRO~lND OF TIIE T~V:~ION
Obesitsr and Li~id Metabolism Disorders Bodv Fat Store5 In humans obesity can be defined as a body weight exceeding 20~ of the desirable body weight for individuals of the same sex, height and frame (Salans, L.B , in E
crinoloqv & Metabolism, 2d Ed., McGraw-Hill, New York 198~, pp. 1203-1244; see also, R.H. Williams, Textbook Qf En~-crinoloqv, 1974, p~. 904-916). In other animals= (or also in humans) obesity can be determined by body weight pat-terns correlated with prolactin profiles given that members of a species that are young, lean and '~healthy" (i.e., free of any disorders, not just metabolic disorders~ h~ave daily plasma prolactin level profiles that folrow a regular pattern with little or no standard deviatio~. - ==
Obesity, or exce~s~fat deposits, correlate with and may trigger the onset of various lipid metabolism disorders, e.g. hypertension, Type II diabetes, atheroscle-rosis, etc.
Even in the absence of clinical obesity ~accord-ing to the above definition) the reduction of==body fat stores (notably visceral fat stores) in man especially on a long-term or permanent basis~would be of significant benefit, both cosmetically and physiologically.
The reduction of body fat stores in domestic animals (as well as pets) especially on~ a~ long-term:=or permanent basis would also obviously be~ o~f considerable economic benefit to man, particularly since farm animals supply a major portion of man's~diet; and the animal fat may end up as ~e novo fat deposits in man.
Whereas controlled ~iet and exerci~se can proauce modest results in the reduction of body fat deposits, prior to the cumulative work of the present inventors (including ~096/00396 2 1 9 2 8 5 5 }~ ',.g ~ ~1 the prior co-pending patent applications and issued U.S.
patents referred to below), no truly effective or practical treatment had been found for c~ntrolling obesity or other lipid metabolism disorders. = == ~
Hyperlipoproteinemia is a condition in which the concentration of one or more of cholesterol- or triglycer-ide-carrying lipoproteins (such as chylomicrons, very low density lipoproteins or VLDL and low-density lipoproteins or LDL)~in p~lasma exceeds a normal~limit. This upper limit is generally defined as the ninety-fifth percentile of a random population. Elevated levels of these substances have also been positively correlated with atherosclerosis and the often resulting cardiac infarction, or "heart attack", which accounts for approxima~tely one-half of all deaths in the United States. Strong clinical evidence has been presented which correlates a reduction in plasma lipoprotein concentration with a reduced risk of athero-sclerosis (Noma, A., et al., Atherosclerosis 49:1, 1983;
Illingworth, D. and Conner, W., in EndocrinoloqY & Metabo-lism, McGraw-Xill, New York 1987). Thus, a significant amount of research has been devoted to finding treatment methods which reduce levels of plasma cholesterol and triglycerides.
Another subset of the ~lasma lipoproteins found in vertebrates are high density lipoproteins, or HDL. HDL
serve to remove free cholesterol=from the plasma. A high HDL conce~tration as a percentage of total plasma choles-terol has been associated with a reduced risk of athero-sclero~is and heart disease. Thus HDL are known in the lay press as "good" cholesterol. Therefore, therapeutic strategies involve attempts both to reduce plasma LDL and VLDL content (that is, reduce total plasma cholesterol), and to increase the XDL fraction of total plasma cholester-ol Several lines of research indicate that si~ply in-creasing HDL is of benefit even in the absence of LDL or VLDL reduction: Bell, G.P. et al., Atherosclerosis 36 47-54, 1980; Fears, ~., Biochem. Pharmacol, 33:21g-228, 1984;

W096/00396 2 1 9 2 8 5 5 P~ ~ s ~

Thompson, G., sr.~Hear~ J. ~I 585-~a8, 1989; slackburn, H.
N.E.J.M. 309:426-428, 1983.
Current therapies -for hyperlipoprot~in~m'~s include a low fat diet and:elimination ~of aggravating factors such as sedentary lifestyle. If the hyperlipoprot-einemia is secondary (i.e. incident to e g. a deficiency of lipoprotein lipase or LDL receptor, -various~~endocrine pathologies, alcoholism, renal disorders, hepatic disor-ders) then control of the underlying disease is also central to treatment Hyperlipoproteinemias are also treated with drugs, which usually alter~the levels of particular components of the total plasma cholesterol, as well as reduce the total plasma~lipid component. Among~the recently introduced drugs to treat hyperlipoproteinemia is lovastatin (MEVACOR~) which selectively inhibits an enzyme involved in cholesterol productIon, 3-hydroxy-3-methylglut-aryl coenzyme A (HMG-CoA) reductase. This drug specifical-ly reduces total cholesterol and can cause a modest (5-10~) increase in HDL concentrations. However, benefits from these therapies vary from subject to subject.
M~ V~L7 use of the HMG-CoA en~yme inhibitor is sometimes accompanied by side effects such as liver toxici-ty, renal myoglobinuria, renal shutdown, and lenticular opacity. The risk~of such side~effects necassita~es close monitoring of the patients (e.g., liver=function is tested monthly).
Another drug prescribed against hyperlipoprotein-emia is clofibrate. The effectiveness of clofibrate also varies from subject to subject and its use is often accom-panied by such side effects as~nephrotic syndromes, myal-gia, nausea and ah~o~;n~l pain.8 5 Diabetes Diabetes, one of the most insidious of the major diseases, can strike suddenly or Iie undiagnosed for years while attacking the blood vessels and nerves. Diabetics, as a group, are far more often afflicted with blindness, heart disease, stroke, kidney disease, hearing 1088, W096/00396 21 92855 P~,lU ,.
~ 5 gangrene and impotence. One third of all visits to physi-cians are occasioned by this disease~and its complications, and diabetes and its complications are a leading cause of untimely death i~ the ~nited ~tates and in the Western world.
Diabetes adversely af~ects the way the body uses sugars and starches which, during digestionr are converted into glucose Insulin, a hormone produced by the pancreas, makes the glucose available to the body~s cells for energy.
Im muscle, adipose (fat) ~nd connective tissues, insulin facilitates the entry of glucose into the cells by an action on the cell membranes The ingested glucose is normally converted in the liver to ~o~ and ~O (5Q~); to glycogen (5~); and to fat (30-4Q~l, the latter being stored in fat depots. Fatty acids from the adipose tissues are circulated, returned to the liver fo~ re-synthesis of triacylglycerol and metabolized to ketore bodies for utilization by the tissues The fatty acids are also metabolized by other organs. Fat formation is a major pathway for carbohydrate utilization.
The net effect of insulin-is to promote the storage and use of carbohydrates, protein and fat. Insulin deficiency is a common and serious pathologic co~dition in man. In insulin-dependent (IDDM or Type I) diabetes the pancreas produces little or no insulin, and insulin must be injected daily for the survival of the diabetic. In noninsulin-dependent (NIDDM or Type II) diabetes the pancreas retains the ability to produce insulin and in fact may produce higher than normal amounts of insulin, but the amount of insulin is relatively insufficient, or less than fully effective, due to ~Pll~ r resistance to insulin.
In either form of diabetes there are widespread abnormalities. In most NIDDM subjects, the fundamental defects to:w~ich the abnormalities can be tr~ced are (1~ a reduced e~try of glucose into various ~peripheral~ tissues and (2) an ;n~r~A~ liberation of glucose into the circu-lation from the liver. There is therefore an extracellular W096/00396 21 928~5 }_I/U~

glucose excess and an intracellular glucose de~iciency.
There is also a decrease in the entry of amino acids into muscle and an increase in lipolysis. Hyperlipoproteinemia is also a complication of diabetes. The cumulative ef~ect of these diabetes-associated abnormalities is se~ere blood vessel and nerve damage. ~
No effective treatment has been found for con-trolling either:hyperinsulinemia or insulin resistance prior to the work of the present inventors. Hyperinsuline-mia is a higher-than-normal level of insulin in the blood.
Insulin resistance:can be defined as a state in which a normal amount of insulin produces a subnormal biologic response. In insulin-treated patients with ~iabetes, insulin resistance is considered to be present whenever the therapeutic dose of insulin exceeds the secretory rate of insulin in normal persons. Insulin resistance is also associated with higher-than-normal levels of insulin i.e hyperinsulinemia -= when normal or elevated level~ of blood glucose are present.
The principal unit of biological time measure-ment, the circadian or daily rhythm, is present at all levels of organization. Daily rhythms have been reported for many hormones inclusive of the adrenal steroids. e.g., the glucocortico- steroids, notably cortisol, and prolac-tin, a hormone secreted by the pituitary. In an early article, discussing the state-of-the-art at that time, it was reported that "Although correlations have been ~ade between hormone rhythms and other rhythms, there is little direct evidence that the time of the daily presence or peak level of hormones has important=physiological relevance.~
See Tem~oral SYnerqism of Prolactin and A~ren~l Steroi~ by Albert H. Meier, ~eneral and Comparative Endocrinology.
Supplement 3, l9~~ Copyright 19~2 by Academic Press, Inc.
The article reports that the peak concentration of prol~c-tin occurs at different times of day in lean and fat animals. The article then describes~avian physiological responses to prolactin injections given daily for several W0 96100396 2 ~ 9 2 ~ ~ 5 ~ Sl ~ 7 days. These responses include increases and decreases~in body fat stores, dependent on the ~ime of.day of the injection_ Eurthermore the time of day when prolactin injections promote loss of body fat coincides with the time o~ day when prolaotin is greatest in lean birds Addition-ally, the.time when prolactin injections promote gain of body fat coincides with the time when prolactin is greatest in obese birds. Prolactin was thus found to stimulate fattening only when injected at certain times o~ the day, and time of the response to prolactin was found to differ between lean animals and fat animals.
In an article titled ~;rc.-~iAn and Seasonal Variation of Plasma Insulin and Cortisol Concentrations in the Svrian Hamster, Mesocricetus Auratus by Christopher ~_ de Souza and Albert H. Meier, Chronobiology International, Vol. 4. No. 2. pp 141-151, 1987, there is reported a study of circadian variations of plasma insulin and cortisol concentrations in scotosensitive and scotorefractory Syrian hamsters r-;nt.;n~ on short and long periods of daylight to determine possible seasonal changes in their daily rhythms. The baseline concentration of insulin was found to be higher in female than in male scotosensitive hamsters on short daylight periods. These differences, it is reported, may account for the observed heavy fat stores in female hamsters kept on short daylight periods. The plasma concentrations of both cortisol and insulin varied through-out the day for the groups of animals tested, but were not equivalent. The circadian variation of insulin and corti-sol di~fered markedly with sex, seasonal condition and day length. Neither the daily feeding pattern or glucose concentration varied appreciably with seasonal condition, or daylight. The time of day, or the season, it is report-ed, do not appear to a~~ect_the ~nn7~ntrations in glucose levels. It is postulated that the daily rhythms of cortisol and insulin are regulated by different neural pacemaker systems, and that changes in the phase relations of circa-dian systems account in part ior seasonai changes in body W096/00396 2 1 9 2 ~ ~ 5 F~~

fat stores. ~he circadian rhythms of prolactin and the glucocorticosteroid hormones, e.g., cortisol, ~ave t'hus been perceived as having important though far ~from fully understood roles in regulating daily and seasonal changes in body fat stores~and in the organization and integration of total animal metabolism. See ~;rça~;~n Hormo~e Rh~thms in ~i~id Requlation by Albert H. Meier and John T. Burns, Amer. Zool. 16-649-659 (1976) : :
In our prior co-pending U.S. Patent Application Serial NoS 192,33~, filed May 10, 1988 ~~we have disclosed and claimed methods for resulating lipid metabolïsm di-sor-ders by administering prolactin (or both prolactin and a glucocorticosteroid ("G~ into the bloodstream of an animal or human on a timed daily basis in an=amount and for a period of time sufficient to modify and reset'the neural phase oscillation of the prolactin daily rhythm which then increases insulin sensitivity. The prolactin (or,prolactin and glucocorticosteroid) injections are~timed to' create a peak in the subject's daily prolactin= (or bot~prolactin and glucocorticosteroid~ profIle that ~coinci:des in time with the peak prolactin (or prolactin and ~GC peaks, respectively) of a lean, insuIin=sensitive human to in-crease insulin sensitivity and reduce body fat stores.
Injections of the same agent(s) are timed towards the peak prolactin time of an obese subject to achieve fat gain,' if desired.
In our cQ=pending prior United States Patent Application Serial No. 463,327,:filed on January 10, ~990 (and its continuation-in-part Serial No.~07~813,I25, fi'led on December 23, lg91 '(publishea~aa'W0 93/12793 on July 8, 1993)) we have disclosed and c~aimed a method of modifying and resetting the neural phase oscillations of:the brain which controls both prolactin and GC in an obese animal (or human) by administering a dopam:ine ago~ist~at a predeter-mined time 'of day such thatIthe prolactin (and/or ~GG) peak(s) of the o~ese animal ~or human) will :be phase-shifted~to occur at the time that it occurs Ithey occur) in W096l00396 2 1 9 2 8 5 5 I ./~ . , ~ g a lean animal (or human), with the result that at least one of body fat stores, body weight, hyperinsulinemia, or hyperglycemia will be reduced and~or insulin sensitivity will be increased In our-co-pending prior United States Patent Application Serial No 719,745, filed June 24, 1991 (pub-lished as ~O 93/00092 on January 7, 1993~ we have disclosed and claimed ~nh~nr~ methods for modifying and resetting the neural phase oscillations of the brain which control prolactin levels comprising both (a) administering to the subject a ~p~minP agonist just a~ter the time at which the normal prolactin profile peaks to reduce prolactin levels to the low ~'day'~ levels and ~b) administering to the subject a prolactin stimulator at a time just before the prolactin level peaks in normal subjects with the objective of causing the subject~s prolactin ~rofile to mimic in shape and time the profile o~ a lean human not s~ffering from one or more of aforementioned metabolic disorders.
United States Patent Application Serial Mo 719,745, also discloses and claims the further administra-tion of ~ thyroid hormone to subjects that are being treated with the dopamine agonist and prolactin stimulator, especially to those subjects that are chronically or seasonally hypothyroid.
In co-pending United States Patent Application Ser No. 995,292, filed on December 22, 1992 (International Application Number PCT/US93/12701) we have disclosed and claimed certain improved methods for diagnosing aberrant prolactin rhythms, determining adjustments to be made to abnormal prolactin rhythms, and normalizing abnormal prolactin rhythms In co-pending United States Patent Application Serial No 171,897, ~iled on ~ecember 22, 1993 ~Interna-tional Application No PCT/US94/14994~ we disclose and claim an accelerated release composition ~r~nt~ining a prolactin inhibitor that is rluickly released into the blood stream of the subject W096/00396 2 1 ~ 2 8 5 ~ r~ 03~l In co-pen~ing United States Patent Application Serial No. 178,56g, filed on January 07, 1994 (Internation-al Application No. PCT~US95/006~3~ we disclose and claim a method of treating various lipid metabolism disorders involving the synergistic ~ombination of diet and the administration of a prolactin inhibitor. ~ ~
Various aspects of the present inventio~ have not been claimed in the foregoing applications. In addition, various i~.-pl~V! ts to and advantageous refinements of the administration protocol and its determination have now been made which increase the effe~tivenes6 of the treatment_ SnMM~RV OF TW~ INV~NTIQN
One aspect of this invention relates to a method for normalizing the daily prolactin profile of a first vertebrate subject in need of such treatment which compris-es the steps of comparing the prolactin profile of the first vertebrate subject to the prolactin profile of other verte-brate subjects having a normal prolactin profile~ and adjusting the prolactin profile of the first vertebrate subject to cause the profile of said first subject to generally conform to (or simulate) the prolactin profile of said second subjects.
A variation of this~aspect :of ~the =invention involves (a) comparing only a set of at ~east two ~prefera-bly at least 4, typically 4-6) prolactin levels of the first subject measured at time points during at least two (preferably at least three) key lntervals of the day to the corresponding prolactin levels of healthy subjects at the same time points during the day, and (b) adjusting the prolactin levels of the first subject: to conform to ~or simulate) the corresponding healthy prolactin levels.
As an e~ample, prolactin levels of the first subject can be measured at least twice during each of an early morning key interval and an early evening key inter-val and preferably at least one or preferably at least two ~VO 96100396 2 ~ 9 2 8 5 5 I ~ 5 1 ~ ~1 ~ 11 additional prolactln levels may be measured during a night interval A second aspect o~ this invention relates to a method for evaluating the daily prolactin profile of a human subject, which comprises the~steps of:
(a) comparing the prolactin profile of~ said subject, said profile having been determined over a contin-uous 24-hour period, to a predetermined standard prolactin profile for healthy human subiects; and (b) determining whether said~human subject has an abnormal daily prolactin profile by ascertaining whether one of the following obtains: (i) at any time point during waking hours the subject's prolactin level is higher than l Standard Error of the Mean (SEM) above the mean prolactin level of healthy subjects at the same time point (or of a standard waking hours mean prolactin level or a standard prolactin profile); and (ii) at any time point during sleeptime the subject~s prolactin level is lower than 1 SEM
below the mean prolactin level of normal (healthy) individ-uals at the same time point.
Preferably, the time points compared will be actual prolactin measurement points (i.e. the prclactin levels of the subject being tested will be blood prolactin levels and not extrapolations) and, most preferably, the deter~in~ti~n will be based on two prolactin measurement points, instead of just one; alternatively, the determina-tion will be preferably based on~ wh~ether one measured prolactin level is higher than 2 SEM during waking hours, or lower than 2 SEM during sleeptime.
A variation of this seco~ aspect of the inven-tion involves: (a) comparing only a set of at least two prolactin levels of said~human (measured:at predetermined time points during each of at least two key intervals of the day) to the corresponding prola~ctin levels (e.g. in a prolactin profile) for healthy humans at the same time points during the day; and (b) determining whether said human subject has an abnormal prolactin profile by ascer-W096/00396 2 1 q 2 8 5 5 taining whether one of the ~ollowi~g obtains: ~i) at~any of said time points that fall during waklng ~ours,~the subject~s prolactin level is higher than 1 SEM above~the corresponding mean prolactin level of healthy subjects=~or above the corresponding mean level of a standar:d~prolactin profile) at the same time point; and (ii) at any of said predetermined time points that fall during sleeptime, the subject's prolactin level is lower than 1 SEM,below ,the corresponding night time level of healthy ~su~jects lor below the correspondlng night~time level of a stanaard prolactin profile) at~the same time point.
A third aspect o~ this invention reIates to a method for determining whether adjustment will be re~uired to "normalize~ or generally conform to or approach an abnormal prolactin profile or level to a standard ~ or healthy) prolactin profile or level, the method=co~mpris:ing:
(a) collecting a plurality of bIood sampIes~from:a, subiect over a c~t;n~l~us time period, the collection of said samples being made at predetermined tim=e intervals (or at predetermined time points) within said time period; E~b) assaying the prolactin content of each of said samples;~(c) plotting the prolactin content of: ea~ck of saia samples against the time at which said sample was collected during said time period to generate a plurality of data points (i.e. correlating the prolactin content with the=time); (d~
generating a prolactin profile by connecti=ng (or=o,therwise fitting a curve through) said data points; and (e~ compar-ing the prolactin profile to a predetermined:normal prolac-tin profile. Steps (cl and (d) ~ogether constitute the step of expressing the prolactin content as a function of time to generate the prolactin profile of the su~ject.:
A variation of this third aspect of the~invention involves measuring only key prolactin levels to generate the data points, omitting step (d), and,:as ~step ~e), comparing each data point to the corresponding,prolac~tin level at the same time point on the predetermined stanaard W096l00396 2 ~ 928 55 r~ 5 ~l .

~ prolactin profile, or to a prolactin level of healthy subjects at the same time point.
A fourth aspect of this~invention relates to a method for determining adjustments that will cause an abnormal daily prolactin profile of a patient to confsrm to or approach a normal indiYidual's daily prolactin profile, which comprises: (a) comparing the prolactin profile of the patient to a predetermined standard prolactin profile for healthy (normal) subjects; (b) detqrmining that at least one of the following obtains: (i) the prolactin level of said patient at any time point (preferably any prolactin measurement points, most preferably any two prolactin measurement points) during waking hours exceeds the corre-sponding prolactin level at the sa~e time point of normal ~healthy) individuals by at least 1 Standard Error of the mean (SEM); and (ii) the prolactin level of.such patient at any time point (preferably any prolactin measurement point, most preferably any two prslactin measurement points) during sleeptime is below the corresponding prolactin level of normal healthy individuals at the same time point by at least one SEM; (c) determining the time at which to admin-ister at least one member selected from the group consist-ing of a prolactin inhibitor and a prolactin stimulator to said subject; and (d) selecting the amount of said prolac-tin stimulator and/or inhibitor to a.djust a subject~saberrant prolactin level so that the subiect~s prolactin profile conforms to or approaches the prolactin profile of normal (healthy) individuals. Again it is preferred that if the determination is based on or,e prolactin measurement point being aberrant, the variation from the normal profile be at l~ast 2 SEM.
A variation sn this four~h ilspect of the inven-tion involves making the determination required in step (b) and the selection required in step (d) based on a compari-son of key prolactin levels of the patient at predeterminedtime points during key intervals of~the day (or night) with W096/00396 2 1 9 2 8 5 5 r~
o the corresponding ~healthy~ or ~normal" prolaotin level~ at the same time points.

3RIEF DESCRIPTION OF T~E ~R~Tt--.C
The present invention is further described with respect to the annexed drawings in which: =~
Fig. 1 is a graph of average ~plasma prolactin levels (nglml~ for healthy individuals (males: "M" curve;
females: "F" curvç) v. time of~day (hours). ~
Fig. 2 is a superimposition~of a~male patient~s abnormal base-level (pre-therapy) prolactin profile (thin dotted grey line) and post treatment improved prolactin profile (thin black line), and the standard normal prolac-tin profile ~thick grey line), all in graph f~orm. (Prolac-tin units are in nanograms per ml, and time of day is in hours.) Fig. 3 is the same type of graphical superimposi-tion as Fig. 2 for another male=patient.
Fig. 4 is an illustrative superimposition ~of various types of typical human aberrant prolactin profiLes (a) - (d).

DET~TT~n DES~RTPTION OF ~ INv~t~TION ~_~
All patents, patent applications:~and literature references cited herein~are in~orporate~ by reference~-in their entirety as if their disclosures were physically present in the present specification. In case of conflict, however, the present disclosure~controls.
It has recently been discovered that metabolic disorders such as the foregoing are associated with aber-rant patterns in the daily levels (and~~fluctuations) ~o~
prolactin and neural oscillators such as those associated with dopamine and serotonin. Healthy (normal) subjects, i.e., lean members of a species not suffering from such metabolic abnormalities have highly predictable daily prolactin profiles, which in humans arç characterized by a low and relatively constant prolactin level during the ~ ~, waking hours ~day) followed by a sharp rise to a peak durirg sleep ~night) and subsequent more gradual tapering down to the waking hours level by mornin~. Conversely, "healthy" individuals have prolactin profiles that are at within 1 SEM of the standard prol~ctin profile M or F of Fig. 1 ~depending on whether they are male or female), preferably for at least two prolactin levels measured at different times or within 2 SEM of the standard prolactin profile tor at least one measured proIactin level.
~ In the present context ~'lean" means not obese and not abnormally underweight. Ir. turn, an obese human is~
defined as a human whose body weight is over twenty percent above the ideal body weight for a given population ~R.H.
Williams, Te~tbook of Endocrinology, 1974, pp. 904-gl6).
An abnormally underweight human is anyone at least 10 below his/her ideal bodyweight. Ideal boay weight (IBW) can be determined by using the Metropolitan Life Insurance Compary standard age/height~weight charts.
A prolactin profile of a~subject is obtained by collPrt;ng blood samples from the subject at timed inter-vals during a consecutive time period (preferably at 1 - 3 hour intervals over approximately a I2 -24 hour period), assaying each blood sample for prolactin content, plotting the time of blood sampling against the r~uantity of prolac-tin present in each sample to generate a data poirt foreach sample, and rnnnPrt; nr- the data points (or other~ise fittirg them into a curve) to form the prolactin profile.
Alternatively (for the vast majority of subjects) a set of only a few prolactin levels~need be obtained by rn11Prt;nr blood samples from the subject at spaced apart times at least once and preierably at least twice during each oi at least two key intervals of a 24-hour period.
One such key interval is early morning (06:00 - 09:00);
another is early evening (16:00 - 20:00~. Spaced apart samples may be collected for ~exampIe at 07:00 and oo:oo during the first interval and at 16:00 and 20:00 during the second interval. It is desirable to collect at least one W096/00396 2 1 9285 5 ~ s additional sample at night, during a third inter~al between 23:00 and 02:00,:~e~g.~at 23:00_or at 24 00 -One or more additional optional~prolactin measurements can be taker:at yet another interval, e.g., a late evening interval from 20:00 - 23:00. Prolactin levels:taken during ~ey intervals will be referred to as ~key prolactin levels"
This alternative method is preferr~d in clinical practice to obtaining 24 hour prolactin pro~iles for several reasons. First, such a procedure r whïch avoids overnight hospital or blood~center stay, is~ sïgnificanfly less costly than 24 hour blood:draws. Second, the reduced number of blood samples drawn is preferred by patients as it is significantly less disruptive to their daily sched-ules. The key prolactin levels are used as indicators of the subject's prolactin profile,~ an~d as~subs~titutes there-of.
Although females generally have higher~prolactin levels and sharper peaks than~maIes, the: shape of ~the normal prolactin profile for both sexes=.ls ~uarltatively similar and does not vary appreciably from no~mal individu-al to normal individual (of the same s~exJ within the same species.
By contrast, individuals who suffer from one or more metabolic disorders have aberrant (often: highly aberrant) daily prolactin profiles ~and levels. These prolactin profiles and levels not only diffe~ substantially from the norm but they can also differ-from one another The vast majority (at least about 30~) of:pa-tients with at least one of the foregoing::metabolic disor-ders have abnormal daily prol:actin~ profiles that ~all generally in one~of four 24-hour patterns represented schematically in Figure 4: :
(a) a flat (peak-free~ high level::(in this context "high" means higher than the "day~ or waking hours prolactin level);

WO9~0039G 17 2 1 9 2 8 5 S

(b) a flat (peak-free). low level (in this context '~low" means as low as or lower than the "day~ or waking hours prolactin level);
(c) a peak during the "day" and a low level at "night" (in this context, "low" means low as compared to the normal average sleeptime p~olactin value between 0100 and 0400~; and (d) a peak during the "day" and a second peak at ~night".
The alternative method of me,asuring key prolactin levels i~ early morning and late e~verlin~ would diagnose patients with abnormalities of type~ra), (c) and (d). If a subject has an abnormal prolactin profile of type (b) above~ that subject would have to give one or two night blood samples which would not avoid overnight stay at a hospital or blood center, "Waking hours" or "day~ means the period of time at which in normal (healthy) humans (not working night shifts or alternate shifts~ prolactin levels are relatively invariant and low (between 07:00 h and 22:Q0 h).
As employed herein the term ~sleeptime~ or ~night" means the period of time which in normal humans prolactin level rises to a peak (between 01:00 and 04:00) and then tapers off The normal average prolactin levels between the hours of 01:00 and 04:00 are between 8.0 and 14.0 nanogram-s/ml for males and between 14.0 and 26.0 nanograms/ml for females. See Figs 1-4.
"Prolactin inhibitor" shall include substances which directly or indirectly inhibit prolactin in a subject (vertebrate animal or human). Nonlimiting examples of prolactin inhibitors include ~prolactin inhibiting ~p~mi n~ agonists such as dopamine and certain ergot-related prolactin-inhibiting compounds Nonlimiting examples of prolactin inhibiting dopamine agonists are 2-bromo-alpha-ergocriptinei 6-methyl-8 beta-carbobenzyloxy-aminoethyl-10-alpha-ergolinei 8-W096t00396 2 1 9 2 8 5 5 r~

acylaminoergolines,:: ~re 6-methyl-o-alpha-(N-acyl)ami~o-s-ergoline and 6-methyl-8 alpha-~N-phenylacetyl)amino-9-ergoline; ergocornine; 9,1~=dihydroergocornlne; D-~-halo-6-alkyl-8-substituted ergolines, e.g., D-2-bromo-6-methyl-8-cyanomethylergoline; and lisuride. Moreover, the non-toxic salts of the prolactin-inhibiting ergot-related compounds formed from pharmaceutically acceptable acids are also useful in the practice of this invention Bromocriptine, or 2-bromo-alpha-ergocryptine, has been found particularly useful in the practice of this inYention.
"Prolactin stimulator" shall include substances which directly or indirectly stimulate prolactin. Nonlimi-ting examples of prolactin~sti~mulators ~include~=dopamine antagonists such as metoclopramide, haloperidol,~pimoziae, phenothi~in~, domperidone, sulpirider chlorpromazine=and serotonin agonists, i.e 7 MAO inhibitors, e:g., pargyline, synthetic morphine analogs, e.g., methadonel antiemetics, e.g., metoclopramide, antipsychotics, e.g., estrogens and others, e.g., tryptophan and 5-hydroxy-tryptophan, mela~to-nin, fluoxitane, and dexfenflllorAminP. Moreover/ the non-toxic salts of the foregoing ~rolactin ~st=imula=ting com-pounds formed from pharmaceutic~ally acceptabie acids are also useful in the practice of this invention. Metoclopra-mide has been found particularly useful in the practice_of this invention.
~ Prolactin modulator' shall refer to either prolactin st;mnl~tmrs, prolactin inhibitors, or both.

DEVEr~PM~NT OF T~E "NORMAL" PROLACTIN PROFI~E AND REY
~EVELS
A statistically significant number of healthy and young (20-35 years of age) humans are selected as follows:
All su~jects are healthy and on normal diurnal work/ rest schedule (nQ night g~uards or~other night-shift workers). They are then divided inEo Ewo different groups according to sex. ~11 subjects must normally sleep betw=een about 23:00 and about 07:00). The i~dividuals are healthy ~W096~0396 2 ~ 9 2 ~ 5 ~ SI
~' 19 in the sense that they are free of physiologic disorders or pathologies. In particular, they are not obese (based on standard age/size/weight tables); are known to have normal plasma levels o~ insulin; and are euthyroid i.e , have normal levels of plasma thyroxin, triiodothyronine, free-thyroxin and TSH, and suffer from no malignancies or autoimmune disorders or gene~ically transmitted diseases.
A statistically signi~icant number shall mean at least 3, the smallest sampling number for which statistical formulas generally have any meaning. However, a sampling number of at least 6 is generally preferred (at least 10 is more preferred) because this amount of sampling generally reduces the standard error (SE) of prolactin determina-tions. :~ ~
Blood is collected from each subject at 1-3 hour intervals over a 24-hour period and, diurnal plasma levels of -prolactin, total triiodothyronine (T3) total thyroxin (T4), free-T4=and thyroid stimulating hormone (TSH) are measured for each subject for e g., blood is collected over a 24-hour period at suitable time intervals (e.g., every 1 to 3 hours), typically starting at lD:00 am and finishing the next morning at 8:00 am).
Suitable sampling techniques and assay procedures are well-known to those skilled in the ~ield and can be selected from pnhli~ procedures, e.g., Linkowski, P. et al., J. Clin. Endocrinol. Metab. 61:429-438, 1985; Van Cauter and Copinschi, "Circadian and Episodic Variations~, Martinus Nyhoff, The Hague Netherlands, pp. 1-25, 1981.
During sampling, all s~bjects must co~sume the same diet and ~;nt~;n the same sleeptime schedule Prolactin data are plotted against time of.day and a normal prolactin curve is developed ' n~ i ng SEM. Mathematical expressions can also be developed to describe the curves and the area under the curves.
The result of a normal 24 hour profile for males approximately 30 years of age is shown as the "M" curve in Figure 1. ~The result for ~emales approximately 30 years of=:

W096/00396 : 2 1 9 2 8 5 5 r~

age i8 the "F" curve. The SEM for prolactin during waking hours for males is l.Q to 2.0 nanograms per ml and:appro~i-mately 3.0 nanograms per ml during sleeptime. For females the SEM for prolactin during waking hours is be~tween l_Q
and 3.0 nanograms per ml. and between 3.0 and 6.0 nanogrxms per ml. during sleeptime.
Alternatively, the curves of Fig. l can be used as the standard prolactin profile (as weIl as for the standard prolactin levels at times corresponding to key prolactin measurements); or only a set of prolactin levels can be measured in healthy subjects at key intervals during the day, e.g., at least two in the early morning,~at least two in the early evening and optionally one or two at night. ~ -_ ~ :
M ~TLON OF DI~RNAL ~ORNONE
PROFILE ~ND/OR KE~ PROLACTIN ~EVELS
OF "AFFECT~n" S~3~EC~S
The procedures described above~can be used to develop a 24 hour prolactin profile (or a set of key levels of prolactin during certain key intervals) for individuals under clinical evaluation for therapy according to tbe pre~ent invention. Individuals that are:expected to have an abnormal daily prolactin rhythm include those having been diagnosed as afflicted with at least one of the ~ollowing conditions: obesity (i.e., more than 20~ over-weight, based on age, frame sl'ze and sex characteristics using tables such as the Metropolitan Life Insurance Company tables for standard weight for height and age~, insulin-resistance, hyperglycemia, hyperinsulinemia, hyperlipidemia, or Type II diabetes. However, the present evaluation is not limited to such subjects.
The subjects to be considered for therapy should be euthyroid, i.e., hav~ a total T3 level between about 9o and about 180 ng/dl, total T4 between about 4.5 and about 12 g/dl, and free-~4 between about 0.7 and about 1.9 g/dl.
If not, T4 and/or T3~ shouId be:administered (preferably in the morning hours) priorrto or concurrently with prolactin ~W096l00396 2 1 ~2 85~ r~"~ c I

~ 21 modulator administration. In other words, it is preferable to adjust the prolactin profile of an=individual if his/her levels of T4 and/or T3 are ~ormal. Thus~ prior to adjust-ing the prolactin pro~ile or lev~ls ~of a subiect through administration of a prolactin stimulator, or inhibitor, or both, it is preferred to determine that the subject is euthyroid. If the thyroid hormone levels in a subject are below normal (as determined with reference to the above normal values) T3 and/or T4 are, preferably, first adminis-tered to the .subject until the plasma levels of thesehormones are normal and prolactin stimulatory or inhibitory treatment is then begun. The thyroid hormone amount administered is the dosage required to bring the patient to a euthyroid condition; usuaIly between about 25 and about 150 mcg per patient per day. If nrrrCc~ry to maintain adequate levels of T3/T4 throughout the period of adminis-tration of prolactin stimulator and~or inhioitor, T3/T4 can be continued along with the ~prol ctin modulator therapy agents. Hyperthyroid subject should also receive treatment (via use of antithyroid agents or thyroid ablation) prior to prolactin modulator treatment.
Once a diurnal prolactin level profile has been developed for an individual, the profiIe is compared to.the "normal" profile (e.g., the one generated as described in the previous section or to Fig. 1). A determination can then be made based on the following general criteria:
1. From about 07:00 h to about 22:00 h, i.e., during the flat and low ~evel portion of the '~normal~
prolactin profile, at a given time,point in the prolactin profile of the subject the (blood) prolactin value must not be higher than one SEM above the mean at the same time point (or higher than 2 SEM above the mean at one time point, or higher than one SEM above the mean at two differ-ent time points). This rule applies to exclude from a conclusion of ~normal" all indivi~ual prclactin profiles that do not meet the above criteria, either because their W096l00396 2 l 9 2 8 5 5 . ~u ~ ~1 prolactin is too high duri~-g the day or because their prolactin is too low during sleeptime, or both.

2. From about 22:0Q h till about 07:00 h, i.e., during the rapid change (and sl eptime peak) of the normal daily prolactin profile, the indlvidual's prolactin profile must first have a peak at about the sa~e time~or within two to six hours after=~sleep initiation as the '~normal~ prolac-tin peak for~subjects in the same category (usually about 01:00) and must aIso be within one SEM of the normal healthy prolactin prQfile (preferably for two prolactin readings or alternatively within two SEM for at least one prolactin reading).~
It should be noted that, based on clini--al experience of the.present inventors, adjusting~high day time prolactin levels to normal or near normal values effects a significant i~ sv~ t in the pathologies of the patients treated even if, as a re-sult of the treatment, the night time prolactin peak is lessened. Therefore., consis-tent with this observation, obser~ance of criterion 1 above takes priority over observance of criterion 2.
If only a set of key prolactin~easurements are made for the subject being tested, the above criteria ~ e applied as follows: if at any of the ''day~ time points at which key prolactin measurements have been made for the subject his/her prolactin level is higher than 1 SEM (and preferably 2 SEM) above the mean day time normal prolactin level at the same time point, then the subject has an abnormal day prolactin profile or rhythm; and if at any ~night" time point for which a key prolastin level has been measured the prolactin level of the subject=-~taken during the key interval of 23:Q0 - 02:00) is lower than 1 SEM (and preferably 2 SEM) below the mean .ileeptime normal prolactin level at the same time point, then the subject has an abnormal night prolactin profile or rhythm.
To determine if a subject has an aberrant prolao-tin profile the bedtime on the subject's prolactin profile should ideally be coincident with the bedtime on the ~W096l00396 2 ~ 9 2 ~ 5 5 r~

profile o~:normal subjects. If this is not the case, the profile o~ the subject and the profile of normal individu-als can be superimposed and one or the other ca~ be shifted so that the sleep initiati~n time of~the subject to be tested coincides with the sleep initiation time of normal healthy subjects.

"~r~ TION OF rRR~TM~T FOR AN AFFECTED SUBJECT
The information ~prolactin profile and set of key prolactin levels) generated as described above is used to (a~ identify the patients that are in need of an adjustment in their prolactin proiile and (b) to ~r~rmi ne the type and extent of adjustment re~uired. In general, those individuals ~hat are obese, hyperinsulinemic, hyperlipid-emic, hyperglycemic and/or diabetic display abnormalprolactin profiles (or key prolactin levels) as compared to healthy individuals. Simply stated, by comparing a subjec-t~ 8 prolactin profile (or key prolactin levels) with the standard prolactin profile, or corresponding healthy (normal) set of prolactin levels, it is possible to identi-fy individuals afflicted with the abnormaL conditions discussed abo~e By adjusting the abnormal prolactin proiile of such individuals ~either by administration of a prolactin ;n~;hit~r or a prolactin stimulator, or both) at the appropriate time of day and in the appropriate dosage (amount) it is possible to adjust such individuals~ prolac-tin profile to~ con~orm (or at least approach) a normal profile. ~he amount and timing of=administration of such dosages can be determined based upon information contained in the prolactin profiles (or key prolactin levels) dis-cussed above. _ An adjusted profile approaches a normal or healthy profile if all or a portion of the abnormal profile moves in the correct direction by at least 2 ng/ml. For example, if a human subject~s abnormal prolactin level is 18 ng/ml between 07:00 and 10:00 and (after adjustment) it is reduced to 16 ng/ml during the same time period, the W096/00396 24 1~~

ad~usted profile approaches~the healthy=profile. It is thus important to reduce the area under the daytime prolac-tin curve (typically by at least about 20~) and to avoid prolactin peaks during the day.
The treatment determination has three~ aspects:
(a) choice of effect desired (i.e., choice of prolactin stimulator to i~crease prolactin levels, or ~prolactin inhibitor to reduce prolactin levels, or-both to increase prolactin at night and to reduce prolactin during the day);
(b~ timing of (each) dose of administration; and Ic) amount of (each) dose to be administered. For example, generally if the patient is initially diagnosed as requiring a prolactin inhibitor, he might initially receive 0 ~-1.6 mgs per day of (pr2ferably) accelerated.-release bromocriptine l'i in a sinyle dose (or in divided doses). The dosage might be adjusted after about 4 weeks Df treatment e.g., at that time a fresh prolactin profile (or a s-et of key prolactin levels) would be taken and e.g., if the patient still had a high prolactin level he would receive an increased dosage of (preferably) an accelerated release prolactin inhibitor at the appropriate time of day. The time Df administratlon might also be adjusted (~r~n~;n~ on the patient's current prolactin profile or ~ey prolactin levels), e.g., the time of bromocriptine administration would be changed.
The comparison of ~the individual prolactin profile (or levels) to the normal prolactin profile (or levels) will determine (a) and ~b) in tEe preceding para-graph, as follows: If at any point during the ~'day~ prolac-tin level is too high (whether or not it has a peak), administration of a prolactin inhibitor is required.
If the "night'~ prolactin profile lacks a peak or if its peak is not sufficiently pronounced, administration of a prolactin stimulator is required.
The preferred prolactin inhibitor (or dopamine agonist) is an accelerated release inhibitor, in particular an accelerated release somposition c~nt~;ning bromocriptine W096l00396 2 ~ ~2855 r~l~u~
~ 25 as set ~orth in co-pendlng U.S. Patent Application Serial.
No. 08/171,897.
The preferred prolactin stimulator is metoclopra-mide.
Whether a 24-hour prolactin profile is generated for a subject to be treated, or only key prolactin levels are measured, the following more specific guidelines will generally be followed to ;niti~lly determine bromocriptine administration timing, for a period of treatment of approx-imately 26 weeks:
a) Week 1 to Week 6. Fir~t ~Qsa~e: If any one of a patient's 07:00, 08:00, 16:00 or~ 19:Q0 prolactin levels is e~ual to or higher than 5.0 ng/ml for males or 7.0 ng/ml for females, then 0.8 mg of accelerated release bromocriptine is administered at 06:00 daily.
Second Doaa~e: Beginning in week 3, a second dosage containing 0.8 mg of accelerated release bromocript-ine is also administered at 10:30 daily.
b) Week 7 to Week 12. First dosaQe: If any one of the oi.00, 08:00, 16:00, or 19:0~ prolactin values is still e~ual to or higher than 5.0 ng/ml for males or 7.0 ng/ml for females, then 1 6 mg of accelerated release ~vllv~iptine are administered at 06:00. Otherwise, Q.8 mg of :accelerated release bromocriptine is administered at 06--00 daily.
Second Do~aae: In addition, if the 19:00 prolac-tin level is less than or eQual to 1.5 ng/ml for males or females then the second dosage of~0.8 mg of accelerated release bromocriptine is administered at 08:30 daily instead of at 10:30. If the 19~ 00 prolactin level is higher than 1.5 ng/ml for males and females, then the second dosage ~nt;nll~q to administered at 10:30 daily.
If the 19:00 prolactin I~vel is less than 1.o ng/ml for males and females, then there is no administra-tion of second dosage.

W096~0396 2 1 92855 ' 26 c) Week 1~ ~o Week 26. For bot~ first and second dosages the rules are the same set forth for Weeks 7 - 12, subject to the following:
(i) If either the 16:00 or l9enO
prolactin level is equal to or higher' than 5.0 ng/ml for males or 7.=0 ng/ml for females, ~hen'a~dd an -ad;tional Q a mg of accelerated; release ~bromocriptine to the first dosage, unless the patient is already receiving 2.4 mg of bromocriptine in total. In that case, add the additional 0.8 mg of accelerated release bromocriptine~to the second dosage;
(ii) If the 19:00 prolactin level is lower than 1.5 ng/ml for males or females, then the second dosage time is adjusted by administering it 2 hours earli-er; and (iii) If each of the oa oo, 16:00 and19:00 prolactin levels is less than 1.0 ng/ml for males or females, then subtract 0.8 mg of accelerated release bromo-criptine from the ~econd dosage, or, if there is no second dosage, then subtract 0.8 mg of accelerated release bromoc-riptine from the ~irst dosage. In the vast majority of patients, the first dosage must contain a minimum of 0.8~mg of accelerated reIease bromocriptine.
The time and amount schedules given above are intended as guideli~es ior bromocriptine administr'ation and those skilled in the art can further adjust the precise timing and amount of bromocriptine administration based on the actual prolactin profile or~key prolactin levels of a patient to be treated For example, if a patient does not respond (or does not respond adequately) to~a given dosage or dosages ~e.g. o.a mg) it (or they) can be increased (e.g. to 1.6:mg).
When needed, metoclopramide ~generally daily dosage range is 0.5 - 5. n mg/person; preferred daily dosage range is 0.5 - 2.0 mg/person) ~can be administered once about one hour be~ore bedtime. A prolactin stimulator in general is not administered un~ess the average prolactin W096t00396 . 2 ~ 92855 r~u~ ~1 .

level o~ the patlent between Ol:OO a~d 04:Qo is at least 1 SEM (preterably based on two data points or.at l~east 2 5EM
if based on only one data point) lower than 8.0 ng/ml for males or 14 ng~ml for females Eurthermore, when the day time prolactin is too high a substantial clinical benefit will be realized by adjustment of the day time prolactin levels and the administration of a prolactin stimulator will not be re~uired in most cases ~although it may be desirable).
In general, the time at which an inhibitory agent other than bromocriptine, or stimulatory agent other than metoclopramide, is to be administered to a patient can be determined by ascertaining the time between administration of the agent and the time at which the agent exerts its maximum biological (i.e., stimulatory or inhibitory) e~fect. The time at which a stimulator has its maximum stimulating effect, (or when an inhibitor has its maximum inhibition effect) can be determined by administering the stimulatory or inhibitory drug to a patient with a known prolactin profile and then calculating the time that elapses between administration of the drug and exertion of the maximum effect on movement leither inkibition or stimulation) of the patient's (known) prolactin profile.
In fine tuning a subject's medication administra-tion schedule, the rebound effect that administration of a prolactin inhibitor during the day might have in sleeptime prolactin levels of this subject should be taken into account. Conversely, the rebound effect that administra-tion of a prolactin sti~ tnr might have on prolactin levels during ~the subject's waXing hours should also be considered ~n this manner, and using the methodology of the foregoing g~ l;nPq, the time of administration for a particular inkibitor or stimulator çan be determined using routine expeYimental procedures.
The precise time of modulator administration that will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon the W096100396 2 1 9 2 8 5 5 r~

activity, pharmacokinetics, and bioavailability of a particular modulator, physi~logical condi~ions ~oi ~he patient (including age, disease~type and~stage, physical condition, responsiveness to a given dosage and modulator~, route oi administration, etc. ~owever, the above guide-lines can be used as the basis for determining the optimum ti~e of administration.
The foregoing are applicable ior setting initial therapy regimens. _In general a patient receives between about 3 and about 100 micrograms of bromocriptine per kilogram of body weight per day, and prefexably between about 10 and 40 mi~,u~la,r,~ per kg of body weight per day.
The exact dosage of: prolactin inhibitor tor prolactin etimulator) required to achieve the optimum effect in terms lS of prolactin adjustmeht must be adjusted for each patient based upon the patient's drug sensitivity (i.e., response to drug) age, disease state and stage and physical condi-tion. The patient is periodical~y reevaluated by measuring prolactin levels at predetermined intervals~during a 24-hour period, preferably (or shorter period if necessary), the first such reevaluation typically occurring at the end of four weeks from the onset of= therapy, and subseguent reevaluations occurring every 4 to 3 weeks during therapy and then every 3 months thereafter. Typical daily dosages of bromocriptine for humans on~a:per patient basis are~0.2 - lS mg, preferably 0.8 - 3 mg.
Prolactin stimulators are noxmally administered within about 1 hour prior-to retiring fox the patient~s normal sleep period.
Adjustments to the amount(s) of~drug(s)~ adminis-tered and possibly to the time of administration may be made as described above based c~ these reevaluations. ~
Generally, adjustment of timing and amount~of drug(s) is not considered, necessary if the sleeptime prolactin peak during therapy is higher than normal as long as the peak value occurs at the right time, and the slopes wo96l003s6 2 1 928 ~5 of the peak are sharp (with normal values at each side of the normal peak).
The efficacy of a particular regimen on a particular patient and the adjustments (in dosage and timing) required, if any, can be determined by comparing the patient's re-ev~ t;rn prolactin profile or reevalua-tion key prolactin levels with the standard profile (or the "healthy~ key profile levels~. ~
In treating vertebrates, generally, dosages of the acc~lerated release prolactin inhlbitor (bromocriptine) and/or stimulator (metoclopramide~, respectively, are each given, generally once a day, generally over a period ranging from about 10 days to about 180 days, but treatment can continue in~Pf;n;tely (if necessary or desired) for months or even years. The preferred prolactin inhibitor (accelerated release bromocriptine) is given daily at dosage levels ranging from about 3 micxograms to about lO0 micrograms, preferably from about 10 micrograms to about 40 micrograms, per~ kg. of body weight, and the prolactin stimulator (metoclopramide) is given daily at dosage levels ranging from~ about 5 micrograms to about 240 micrograms, preferably from about 5 mi~L~yLdrrs to about lO0 micrograms, per kg. of body weight per day to modify, or alter, the prolactin profile (or the key prolactin level) and contin-ued for a time sufficient to reset the circadian plasmaprolactin rhythm, at which time treatment may be discontin-ued. If ~he subject suffers a relapse, treatment may be resumed.
In treating humans, in partic~lar, the prolactin ir~ibitor (accelerated release bromocriptine) is generally given at daily dosage level~ ranging from about 3 micro-grams to about lO0 micrograms, preferably from about 10 micrograms to about 40 micrograms, Fer kg. of body weight.
The prolactin stimulator metoclopramlde is y-enerally given at daily dosage levels ranging from about 5 micrograms to about 50 micrograms, preferably from about 5 micrograms to about 20 micrograms, per kg. of body weight per day. (Per W096l00396 21 92~5 p~ oSI ~

person dally dosages range of metoclopramide are~typlcally 0.5 to 5.0 mg; preferably 0.5 to 2.0 mg.l Such treatment (using one~or both types of drugs) i5 typically~continued over a period o~ time ranging from about 10 days to usu~lly about 180 days, resulting in modification and resetting~of the lipid and glucose metabolism of the patient to that oi a lean (i.e., normal) healthy person, at which time treat-ment may be discontinued. For some patients (e.g. patients in particularly poor physical condition, or those of an advanced age) it may not be possible to reset the~ir prolac-tin rhythm withi~ the above time periods and such patients may require a longer, or even r~nt;nl~rus, treatment with prolactin stimulators and/or i~hibitors ~he dosage and timing informatio~ set forth above is designed for~bromocr-iptine and metoclopramide and will have to be altered for other agents using ~the dosage and timing methodology disclosed herein.
In the practice of this invention,~ a prolactin-inhibiting compound, and a prolactin stimulator are admin-istered daily to a subject preferably orally, or by subcu-taneous, intravenous or intramuscular;iniection:_ Dermal delivery systems e.g., skin patches, as well as supposito-ries and other well-known systems for administration~of pharmaceutical agents can also be employed.
sOdy fat deposits, inclusive of adipose/ arterîal wall and plasma fat, of an obese person will be reduced, leveled out and generally maintained ~(after the treatments of the present invention are disc~nt;nl,r~) at that of a normal (lean) person, over an r~tr~d period of time.~ A
subject that exhibits the effects of insulin resistance, hyperlipidemia or hyperinsulinemia and/or hyperglycemia, or both insulin resistance and hyperinsulinemia andlor hyper-glycemia, treated with the prolactin inhibitor and/or a prolactin stimulator at the ~appropriate times of day discussed~above, will become more sensitive tQ insulin (i.e., will have a lower insulin resistance), and the effects of hyperinsn7;nrmi~ ~and/or hyperglycemia and W096l00396 2 1 9 2 8 ~ 5 r~

related ab~ormal metabolic values will be reduced on a long term basis. Treatment generally lasts between about 10 and about 18D aays on average in humans The administration of the prolactin inhibitor ~or dopamine agonist) and/or prolactin stimulator in this manner will thus reset the phase relations~ of=the two neural oscillations and their various circadian expressions to alter metabolism on a long term basis (e g., several years), if not permanently~ In other words, the result of the timed daily dosages of the prolactin inhibitor ~or dopamine agonist) and/or prolactin stimulator will be a long term reversal of the major pathologies generally associated with the development of Type II diabetes. Using the methods of=the present inven-tion, the levels of body fat stores, plasma insulin concen-trations (including in patients oral hypoglycemic medica-tions), insulin resistance, hyperglycemia, and blood pressure or all of these pathologies can be reduced on a long term basis by such treatmènt, or treatments, from the high levels often found in obese, hyperinsulinemic, hyper-lipidemic and/or hyperglycemic persons to approach or conform to the much lower and much more desirable levels found in normal persons with normal insulin levels.
The following are non-limiting working examples of diagnosis, regimen determination and therapy according to the present invention:
Case StudY 1: ~13001Q) Subject: male; 52 yrs; 294 lbs; 5 ft 6 in.

Pretreatment ~atholoqy: , (a) Obesity: 191~ IBW (based on a standard Table, e.g., the standard table of Metropolitan Bife.Insurance Co.
NY, ~Y available from the company);
(b) Type II diabetes: fasting plasma glucose 164 mg; percent glycosylated hemoglobin 9.4~ (measured by affinity chromatography)i The normal morning fasting plasma glucose is between 80 and 12~ mg percent (mg ~).

W096/00396 2 1 q 2 8 5 5 The subject's 24-hour base (pre-therapy) prolac-tin profile is shown graphically aE the dotted grey line~in Figure 1 It shows that the subject's prolactin~was too high throughout the day and early evening, At 07:00, it was 13.2 ng/ml and at 08:00,-ll~a~ng~ml; ~at 16:~-it was 9 9 ng/ml and at 19:~v0 it was 12'2 ng/ml The subject was administered accelerated release bromocriptine as follows:
Weeks 1 and 2: 1 6 mg at 05:00;'weeks 3 and~4: 0 8 mg at 05:00 and an adaitional o:a mg at'10:0~; ~reeks 5 -~ 18: 1 6 mg at 05:00 A ~~reevaluation)' prolactinl oro'~ile was generated for the subject after 13 weeks and i~s graphically Ehown in Fig 2 as the thin black line_ The ~ey prolactin values were 07:00 11 6 ng/ml; 08:00 9 5 ng/ml; 16:00 3~3 ng/ml and l9:a0 6 5 ng/ml. ~ ~
The i , L~V. ts observed in this patient's prolactin levels after 12 weeks of treatment included significantly lower levels throughout most of the day and early evening The clinical benefits of treatment to this patient included reduction in glycosylated hemoglobin from a starting level of 9 4~ to 7~ 6~,after 18 weeks of treat-ment. The patient lost 18 pounds of body fat'::over 18 weeks; plasma cholesterol was reduced from 229 mg ~ to 180 mg ~; triglycerideE were reduced from 186~mg ~O to,1,,22 mg ~;
fasting glucose was reduced from~164 mg ~ to 133 mg ~; and oral glucoEe tolerance test results improved, over the treatment period Case Studv 2: (130007) Subject: male; 53 yrs; 203 lbos;~5 ft. 1~ in Pretreatment ~atholoqv: : , (a) Obesity: 130~ IBW;
(b) Type II diabetes: fasting pIaEm=a glucose 16a mg ~, glycosylated hemoglobin~7~5~
On initial evaluation, the subject had the prolactin profile shown in Figure 3 aE~ the dotted grey line. The day levels ~particularly the 07:=00 level) are W096l00396 21 9 2 8 5 5 r ~ Sl too high: at 7:00, 18.2 :ng/ml; at 8:00, 15.0 ng/ml; at 16:00, 8.1 ng/ml; and at 19:00, 8.8 ng/ml. The night time peak is also somewhat delayed. The patient was adminis-tered accelerated release bromocriptine as~follows- Weeks l and 2: 1.6 mg at 08-3Q; Weeks 3 and 4: 1.6 mg at 08:Q0;
Weeks 5 and 6: 0.8 mg at 08:30i Weeks 7 - 18: 1.6 mg at 05:00.
The second profile (the thin black line) taken after 18 weeks of treatment shows that the subject~s prolactin profile was very sensitive to bromocriptine. The day time levels were significantly reduced. However, the night time levels were also reduced below normal Prolac-tin levels were as follows: at 7:00, 1.9 ng/ml; at 8:00, 1.7 ng/ml; at 16:00, 0.9 ng/mli and~at 19:0Q, 1_5 ng/ml.
The clinical benefits of treatment to this patient included reduction in glycosylated hemoglobin from a starting level of 7.5~ to 6.1~. The patient lost 23 pounds of body fat; the patient's OGTT test showed consid-erable improvement, with the area under the glucose curve declini~g by approxi~ately 30~; and fasting glucose was reduced from 168 mg ~ to 133 mg ~, over the course of treatment.
The data show that metabolic states are regulated at least to a clinically significant degree in part by an interaction of circadian neuroendocri~e rhythms. This hypothesis proposes that the daily rhythms of cortisol and prolactin are individual expressions of two separate circadian systems and that the phase relations of these two systems can be reset. Thus, in a hamster model it has been found that the 0-hour relation resets the circadian oscil-lations into a pattern that maintalns the lean, insulin sensitive state and the 12-hour relation permits retention of a pattern that maintains the obese, insulin resistant state. Another important addition of the present study is that the effects:o~ timed injections of a prolactin inhib-iting dopamine agonist, or other prolactin inhibiting compound, are long lasting. Apparently once resetr the W096/00396 2 ~ 92~55 ~ J5 ~l 34 ~
phase relation ~f the_t~o clrcadian oscillations~ tends ~o maintain its altered pattern Changes in the phase relations oi two~circadian neuroendocrine oscillations are eYidenced ~y changes in the phase relations of their ~ir~ n expressio~s This expectation is fulfilled respecting plasma glucocorticost=e-roid and prolactin rhythms. In=several species~examined~
the phase relations of the two hormone rhythms differ:~in lean and fat animals.
The phase relation between the circadian rhythm of plasma insulin concentration and the~rhythm of lipogenic responsiveness to.insulin is shown to differ in=lean and fat animals. Whereas the daily interval of :lipogenic responsiveness remains near light onset, the phase of the insulin rhythm varies markedly. The peak concentration of insulin, e.g., occurs ~ear light onset =in obese female hamsters held on short day-lengths. Tha=t is, the daily peaks of the lipogenic stimulus (i e., insulin) and the lipogenic response~to insulin co~incide in fat animals and not in lean animals.
It is apparent that various modifications and changes can be made without departing from the spirit and scope of this invention.

Claims (31)

WHAT IS CLAIMED IS:

1. A method for determining abnormalities in the daily prolactin rhythm of a first vertebrate subject having a daily prolactin rhythm which comprises the steps of:
a. measuring the blood prolactin level of the first subject at spaced apart intervals during the first subject's waking hours and during the first subject's sleeptime over a time period of about 24 hours to generate a prolactin profile for said first subject;
b. comparing the prolactin profile to a predetermined standard prolactin profile for healthy subjects of the same species; and c. identifying said first subject as having an abnormal daily prolactin rhythm if the first subject's measured prolactin level is (i) is higher than 1 SEM above the mean prolactin level for said healthy subjects at two time points during waking hours; or (ii) lower than 1 SEM
below the mean prolactin level for said healthy subjects at two time points during sleeptime.

2. The method of claim 1, wherein in step (c) the first subject's prolactin level is a prolactin level measured in step (a) and is (i) higher than 2 SEM above the mean prolactin level for said healthy subjects during waking hours; or (ii) lower than 2 SEM below the mean prolactin level of said healthy subjects during sleeptime.

3. A method for determining abnormalities in the daily prolactin rhythm of a first human subject which comprises the steps of:
a. measuring the blood prolactin level of the first subject at spaced apart intervals during the first subject's waking hours and during the first subject's sleeptime over a time period of about 24 hours to generate a prolactin profile for said first subject;

b. comparing the prolactin profile to a predetermined standard prolactin profile for healthy subjects of the same sex; and c. identifying said first subject as having an abnormal daily prolactin rhythm if the first subject's measured prolactin level is (i) higher than 1 SEM above the mean prolactin level for said healthy subjects at two time points during waking hours; or (i) lower than 1 SEM below the mean prolactin for said healthy subjects at two time points during sleeptime.

4. The method of claim 3, wherein in step (c) the first subject's prolactin level is a prolactin level measured in step (a) and is (i) higher than 2 SEM above the mean prolactin level for said healthy subjects during waking hours; or (ii) lower than 2 SEM below the mean prolactin level of said healthy subjects during sleeptime.

5. The method of claim 1 wherein said predetermined standard has been generated for a statistically significant number of healthy subjects of the same sex as said first subject.

6. A method for detecting abnormalities in the daily prolactin rhythm of a group of human subjects being tested which comprises the steps of:
a. comparing a prolactin profile of said subjects being tested that has been compiled over a predetermined period of about 24 hours to a predetermined standard prolactin profile for healthy human subjects; and b. identifying those members of said group who have a blood prolactin level that is (i) higher than 1 SEM above the prolactin level of said healthy subjects at two different time points during waking hours; or (ii) lower than 1 one SEM than the prolactin level of said healthy subjects at two different time points during sleeptime.

7. The method of claim 6 wherein in step (b) said prolactin level of said members is a blood prolactin level and (i) at any point during waking hours is at least 2 SEM higher than the prolactin level of said healthy subjects; or (ii) at any point during sleeptime is at least 2 SEM lower than the prolactin level of said healthy subjects.

8. A method for detecting abnormalities in the daily prolactin rhythm of a human subject being tested which comprises the steps of:
a. comparing a prolactin profile of said subject being tested, said profile having been determined over a period of time of about 24 hours, to a predetermined standard prolactin profile for healthy individuals; and b. determining whether said subject being tested has an abnormal daily prolactin rhythm based on whether the prolactin level of the subject being tested is a blood prolactin level and (i) is higher than 1 SEM above the waking hours prolactin level of healthy subjects at two different time points; or (ii) lower than 1 SEM below the sleeptime prolactin profile of healthy subjects at two different time points.

9. The method of claim 8 wherein in step (b) the prolactin level of the subject being tested is a blood prolactin level and (i) at any time point is higher than 2 SEM above the waking hours prolactin level of healthy subjects; or (ii) at any time point during sleeptime is lower than 2 SEM below the sleeptime prolactin level of healthy subjects.

10. A method for determining abnormalities in the daily prolactin rhythm of a first vertebrate subject having a daily prolactin rhythm which comprises the steps of:
a. measuring the blood prolactin level of the first subject at spaced apart intervals at least twice during early morning and at least twice during early evening to generate a set of key prolactin levels for said first subject;
b. comparing each of said key prolactin levels to a corresponding predetermined standard prolactin level for healthy subjects of the same species at the same time point; and c. identifying said first subject as having an abnormal daily prolactin rhythm if at least one key prolactin level of said first subject during waking hours is higher than 1 SEM above the corresponding standard prolactin level for said healthy subjects.

11. A method for determining abnormalities in the daily prolactin rhythm of a first human subject which comprises the steps of:
a. measuring the blood prolactin level of the first subject at spaced apart intervals at least twice during early morning and at least twice during early evening to generate a set of key prolactin levels for said first subject;
b. comparing each of said key prolactin levels to a corresponding predetermined standard prolactin level for healthy subjects of the same sex at the same time point; and c. identifying said first subject as having an abnormal daily prolactin rhythm if at least one key prolactin level of said first subject-during waking hours is higher than 1 SEM above the corresponding standard prolactin level for said healthy subjects.

12. The method of claim 10 wherein said predetermined standard has been generated for a statistically significant number of healthy subjects of the same sex as said first subject.

13. A method for detecting abnormalities in the daily prolactin rhythm of a group of human subjects being tested which comprises the steps of:
a. comparing a set of key prolactin levels of said subjects being tested that has been compiled over a predetermined period of about 24 hours to a set of corresponding predetermined standard prolactin levels for healthy human subjects at the same time points as said key prolactin levels of said subjects being tested; and b. identifying those members of said group who have (i) at least one key prolactin level during waking hours that is higher than 1 SEM above the corresponding standard prolactin level of said healthy subjects; or (ii) at least one night key prolactin level that is lower than 1 SEM below the corresponding standard prolactin level of said healthy subjects.

14. A method for detecting abnormalities in the daily prolactin rhythm of a human subject being tested which comprises the steps of:
a. comparing a set of key prolactin levels of said subject being tested, to a set of corresponding predetermined standard prolactin levels for healthy individuals at the same time points as said key levels; and b. determining whether said subject being tested has an abnormal daily prolactin rhythm based on whether (i) at least one waking hours key prolactin level of the subject being tested is higher than 1 SEM above the corresponding standard prolactin level of healthy subjects;
or (ii) at least one night key prolactin level of said subject is lower than 1 SEM below the corresponding standard prolactin level of healthy subjects.

15. A method for normalizing the daily prolactin rhythm in a first subject having an abnormal prolactin daily rhythm which comprises the steps of:
comparing a set of key prolactin levels of said subject to a corresponding set of prolactin levels of healthy subjects of the same species at the same time points as said key levels; and adjusting the key prolactin levels of said first subject to generally conform to or approach the corresponding prolactin levels of said healthy subjects.

16. The method of claim 15 wherein said adjusting step comprises at least one of:
administering to said first subject a prolactin inhibitor if at least one key prolactin level of said first subject during waking hours is higher than 1 SEM
above the corresponding prolactin level of healthy subject;
and administering to said first subject a prolactin stimulator if at least one night key prolactin level of said first subject is lower than 1 SEM below the corresponding prolactin level of healthy subject.

17. A method for determining adjustments that will cause an abnormal daily prolactin rhythm of a patient to conform to or approach a healthy subject's daily prolactin rhythm, which comprises:
a. comparing a set of key prolactin levels of said patient to corresponding standard prolactin levels at the same time points for healthy human subjects of the same sex as said patient;
b. determining that said patient should be administered a prolactin inhibitor when at least one key prolactin level of said patient exceeds the corresponding prolactin level of healthy subjects at the same time point by more than 1 SEM;

c. selecting at least one time for administration of said prolactin inhibitor that is between 06:00 h and 12:00 h; and d. selecting the amount or amounts of said prolactin inhibitor to be administered to said subject so that at least one waking hours key prolactin level of said subject will conform to or approach the corresponding prolactin level of healthy subjects.

18. A method for determining adjustments that will cause an abnormal daily prolactin rhythm of a first subject to conform to the daily prolactin rhythm of a healthy subject, comprising:
a. comparing a set of key prolactin levels of said first subject to a predetermined standard prolactin profile for healthy subjects;
b. determining that said subject is in need of a prolactin stimulator by ascertaining that a least one key prolactin level of said first subject during sleeptime is at least 1 SEM lower than the normal prolactin level of said healthy subjects during sleeptime;
c. selecting the timing of said administration to said subject to be prior to the time at which the sleeptime prolactin level reaches its peak in healthy subjects; and d. selecting an amount of said prolactin stimulator to be administered to said subject such that at least one key prolactin level of said subject during sleeptime is no lower than 1 SEM below the corresponding prolactin level of said healthy subjects at the same time point.

19. A method for determining abnormalities in the daily prolactin rhythm of a first human subject comprising:
a. collecting blood samples from said first subject at least two of said blood samples being collected at predetermined spaced apart time intervals during early morning and at least two of said blood samples being collected during early evening;
b. measuring the prolactin content of each of said samples;
c. plotting the prolactin content of each of said samples against the time at which said sample was collected to generate a key prolactin level data point;
d. comparing said key prolactin level data points to a predetermined standard prolactin profile for healthy subjects; and e. identifying said first subject as having an abnormal daily prolactin rhythm based on whether at least one key prolactin data point of said subject is higher than 1 SEM above a corresponding point on the standard prolactin profile of healthy subjects.

20. The method of claim 10 further comprising:
in step a, measuring the blood prolactin level of the first subject at least once at night to generate at lease one night key prolactin level for said first subject;
in step b, comparing said at least one night key prolactin level to a corresponding predetermined standard prolactin level for healthy subjects at the same time point; and in step c, identifying said first subject as having an abnormal daily prolactin rhythm if said at least one night key prolactin level is lower than 1 SEM below the corresponding standard prolactin level.

21. The method of claim 11 further comprising:
in step a, measuring the blood prolactin level of the first subject at least once at night to generate at lease one night key prolactin level for said first subject;

in step b, comparing said at least one night key prolactin level to a corresponding predetermined standard prolactin level for healthy subjects at the same time points; and in step c, identifying said first subject as having an abnormal daily prolactin rhythm if said at least one night key prolactin level is lower than 1 SEM below the corresponding standard prolactin level.

22. The method of claim 10 wherein said prolactin inhibitor is bromocriptine and said subject is a human.

23. The method of claim 22 wherein said bromocriptine is administered at least once between 06:00 and 12:00h.

24. The method of claim 23 wherein said bromocriptine is administered at a total daily amount within the range of 0.8 - 8.0 mg.

25. The method of claim 24 wherein at least a portion of said bromocriptine is administered at 6:00 AM.

26. The method of claim 16 wherein said prolactin inhibitor in bromocriptine and said subject is a human.

27. The method of claim 26 wherein said bromocriptine is administered at least once between 06:00 and 13:00h.

28. The method of claim 27 wherein said bromocriptine is administered at a total daily amount within the range of 0.8-8.0 mg.

29. The method of claim 28 wherein at least a portion of said bromocriptine is administered at 6:00 AM.

30. The method of claim 10, wherein in step (c) the first subject's key prolactin level is (i) higher than 1 SEM above the corresponding standard prolactin level for said healthy subjects at two time points during waking hours.

31. The method of claim 10, wherein in step (c) the first subject's key prolactin level is (i) higher than 2 SEM above the corresponding standard prolactin level for said healthy subjects during waking hours.