Method of alleviating diseases by cell-mediated immune modulation

Method for alleviating diseases in animals susceptible to treatment via stimulation of the cell-mediated immune system and compositions useful therefor.

BACKGROUND OF THE INVENTION 
(a) Field of the Invention 
This invention relates to a method for alleviating diseases in animals 
which are susceptible to treatment by stimulation of the cell-mediated 
immune response system comprising administering to a diseased animal a 
1-substituted-pyrrole derivative and to compositions useful therefor. 
(b) Description of the Prior Art 
The use of immuno modulators which affect the immune response system in 
such a way as to combat diseases in animals which are susceptible to 
treatment by immuno modulators is well known. For example, bacterial or 
viral infections, such as Staphylococcus aureus and Herpes simplex virus, 
Type 2, (HSV2), have been successfully treated with levamisole 
[(1)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole; Fischer et al., J. 
Infect. Diseases, 132, 578-581 (1975)]. The remission or control of 
certain forms of cancer has been achieved with BCG vaccine (Bacillus of 
Calmette and Guerin, a strain of Mycobacterium bovis) [Holmes et al., 
J.A.M.A., 232, 1052-1055 (1975)], an immunologic adjuvant, or with 
levamisole [Brit. Med. Jour., 3, 461-464 (1975)], whose immuno stimulant 
properties in other areas is well known as indicated above. Levamisole has 
also been shown to have some effect in the treatment of rheumatoid 
arthritis by stimulation of cell-mediated immunity [Huskisson et al., The 
Lancet, I, 393-395 (1976)]. 
Other immuno stimulants disclosed by the art are: "a . . . water-soluble 
substantially neutral polymer of acrylic acid cross-linked with . . . 
polyalkyl-sucrose or polyalkylpentaerythritol . . . " 
[Lund, U.S. Pat. No. 3,920,811, patented Nov. 18, 1975]; a nucleotide or 
complex of polynucleotides [Hilleman et al., U.S. Pat. No. 3,906,092, 
patented Sept. 16, 1975]; and 
"a macromolecular synthetic resin complexing material such as an acrylic 
acid polymer cross-linked with a polyalkyl saccharide . . . " 
[Glass et al., U.S. Pat. No. 3,919,411, patented Nov. 11, 1975]. 
The 1-substituted-pyrrole derivatives used in the practice of this 
invention having the formulas I, II, III and IV below have been disclosed, 
together with their in vitro antibacterial activity in Belgian Pat. No. 
816,542, and each of the compounds of Formulas I, II, III, IV and V, and 
the anti-tubercular activity of two of them, i.e. Compounds I and III, 
have been disclosed in Johnson U.S. Pat. No. 4,051,147, as well as in 
divisional application thereof, Ser. No. 730,162, filed Oct. 7, 1976, and 
now U.S. Pat. No. 4,138,405. Compounds I, II, III and V are claimed in 
U.S. Pat. No. 4,138,405, and Compound IV is claimed in U.S. Pat. No. 
4,051,147. The compounds are disclosed in both U.S. Pat. No. 4,051,147 and 
U.S. Pat. No. 4,138,405 to be useful as in vitro antibacterial agents 
against a variety of microorganisms. Two of the species (Compounds I and 
III) are also there disclosed to be effective against systemic 
Mycobacterium tuberculosis infections on oral administration, and 
Compounds III, IV and V are further there disclosed to be useful as 
urinary antiseptic agents on oral administration in mice. 
However, there has been no indication or suggestion in the prior art that 
Compounds I, II, III, IV, and V have immuno stimulating properties. 
Natural cellular immunity in the normal host is known to be initiated by 
contact between an invading foreign antigen (bacteria, virus, protozoa, 
neoplastic cell, etc.) and thymus-derived lymphocytes (T-cells) which, 
when stimulated by foreign antigen, release soluble factors (lymphokines) 
into circulation. These factors or enzymes produced by the T-cells in turn 
activate the macrophages which destroy the invading organism by a process 
of phagocytosis followed by a direct attack on the organism by enzymes 
that dissolve the invading organism (lysosomal enzymes). Organisms such as 
tubercle bacilli and leprosy bacilli survive phagocytosis and even 
multiply within nonactivated macrophages. The activated macrophage however 
destroys these organisms by increased concentrations of lysosomes and 
lysosomal enzymes [Bennett et al., J. Transplantation, 5, 996-1000]. 
When the normal host is exposed to overwhelming numbers of invading 
organisms or its immune response system has been compromised by a 
deficiency in the immune system, only immune stimulation from sources 
outside the host can prevent a takeover by the intruder. 
In the case of alleviation of allergic or inflammatory diseases, diabetes 
or essential hypertension, the role of immuno stimulators is less clear. 
Inflammatory conditions, such as rheumatoid arthritis, are characterized 
by proliferation in the affected joints of lysozomal enzymes possibly 
produced in the phagocytic process by the macrophage cells. Alleviation of 
the inflammatory condition may, in certain conditions, involve 
suppression, rather than stimulation, of the immune system by the immuno 
modulator. Thus the same chemical entity may appear to exhibit immuno 
stimulation as well as immuno depression of the immune response system, 
depending upon the particular disease condition being alleviated, and it 
may be more accurate to refer to such entities as immuno modulators rather 
than immuno stimulants or immuno suppressants, since they are usually 
capable of behaving in both capacities. (See for example the references 
given above with respect to use of levamisole as an immuno modulator.) 
Chemically induced diabetes in mice or rats has been associated with 
impairment of cell mediated immunity (Brown et al., T-Cell Function in 
Diabetic Mice, [Paper No. 5239] Proc. Ann. Meeting Fed. Am. Soc. Exp. 
Biol. and Med., Chicago, Ill., 1976). This involvement of cellular 
immunity or the modification of the disease by immuno modulation is not 
only unexpected but unexplainable with our present knowledge. 
SUMMARY 
This invention relates, in a method aspect, to a method of alleviating 
diseases in animals which are susceptible to treatment by stimulation of 
the cell-mediated immune response system comprising orally administering 
to a diseased animal an effective amount of 
1,1'-(1,4-benzoquinon-1-yldiimino)dipyrrole, 
1,1'-[(1,4-phenylene)diamino]dipyrrole, 
1-(1,4-benzoquinon-1-ylimino)pyrrole, 1-(4-hydroxyphenylamino)pyrrole or 
1-(phenylamino)pyrrole. 
In a composition of matter aspect, the invention relates to compositions 
for alleviating diseases in animals which are susceptible to treatment by 
stimulation of the cell-mediated immune response system which comprises 
1,1'-(1,4-benzoquinon-1,4-yldiimino)dipyrrole, 
1,1'-[(1,4-phenylene)-diamino]dipyrrole, 
1-(1,4-benzoquinon-1-ylimino)pyrrole, 1-(4-hydroxyphenylamino)pyrrole or 
1-(phenylamino)pyrrole in a pharmaceutical carrier.

DETAILED DESCRIPTION INCLUSIVE OF THE PREFERRED EMBODIMENTS 
More specifically, this invention provides a method for alleviating 
diseases in animals which are susceptible to treatment by stimulation of 
the cell-mediated immune response system which comprises administering to 
a diseased animal an effective amount of 
1,1'-(1,4-benzoquinon-1,4-yldiimino)dipyrrole (Compound I), 
1,1'-[(1,4-phenylene)diamino]-dipyrrole (Compound II), 
1-(1,4-benzoquinon-1-ylimino)pyrrole (Compound III), 
1-(4-hydroxyphenylamino)pyrrole (Compound IV) or 1-(phenylamino)pyrrole 
(Compound V) having the respective formulas: 
##STR1## 
It has been surprisingly found that Compounds I, II, III, IV and V are 
effective in alleviating a variety of diseases in animals which are 
susceptible to treatment by stimulation of the immune response system. 
This discovery indicates usefulness of the compounds of the invention 
against a variety of animal diseases which are susceptible to treatment 
with immuno stimulants, including diseases of bacterial origin, such as 
leprosy caused by Mycobacterium leprae; diseases of viral origin, such as 
Herpes infections; diseases involving inflammatory conditions, such as 
rheumatoid arthritis, allergic encephalitis, lupus erythematosus, Masugi 
nephritis or Crohn's disease; diseases involving allergic reactions, such 
as allergic asthma, diseases involving protozoal infections, such as 
toxoplasmosis and leishmaniasis; and diseases of unknown etiology in which 
cell mediated immunity has been suggested or described, such as diabetes, 
essential or spontaneous hypertension, various neoplastic diseases and 
multiple sclerosis. 
Diseases of many of these types have been treated with varying degrees of 
success with immuno stimulants. See for example Blanden et al., J. Exp. 
Med., 129, 1079 (1969) [treatment of Listeria monocytogenes infections 
with BCG]; Gaugas et al., Nature, 219, 408-409 (1968) [treatment of 
leprosy]; Fischer et al., Pediat. Res. 8, 1974 [treatment of brucella 
infections]; Kint et al., New Eng. J. Med. 291, 308 (1974) [treatment of 
Herpesvirus labialis infections]; Huskisson et al., The Lancet, I, 393-395 
(1976) and Schuermans, The Lancet, I, 111 (1975) [treatment of rheumatoid 
arthritis]; Symoens et al., Brit. Med. J. IV, 592 (1974) [treatment of 
aphthous stomatitis]; Bertrand et al., Nouv. Presse med., 3, 2265 (1974) 
[treatment of Crohn's disease]; Fischer et al., J. Infect. Diseases, 132, 
578-581 (1975) [treatment of protozoal infections]; and Holmes et al., 
J.A.M.A., 232, 1052-1055 (1972), Study Group for Bronchogenic Carcinoma, 
Brit. Med. Jour. 3, 461-464 (1975), and Carter, Amer. Sc., 64, 418-423 
(1976) [treatment of cancer] for examples of the use of various immuno 
stimulants in the treatment of diseases of the above-indicated types. 
Moreover, multiple sclerosis, a disease in which the myelin sheath that 
insulates the nerves of the brain and spinal chord is attacked by the 
individuals own macrophages and antibodies, is an outstanding example of 
diseases caused by deficiencies in the cell mediated immune system. Such 
diseases may also be influenced by the immune modulation activities of the 
subject compounds. 
In any event, with a knowledge of the mechanisms involved in modulation of 
the immune response system as described in the Description of the Prior 
Art section above at hand, compounds which owe their activity against 
disease conditions to their ability to modulate the immune response system 
can be distinguished from compounds which owe their effectiveness to a 
direct action on a pathogenic microorganism, for example, by use of 
experiments designed to test the behavior of the compounds against any of 
the above-indicated parameters involved in the immune response system. 
Thus the immuno modulator properties of the compounds of the present 
compositions have been established by the following experiments. 
(1) Although Compounds I and II are equally active in vitro against the 
Mycobacterium tuberculosis H37Rv organism (3.1 mcg./ml.), Compound I is 
very effective against systemic mouse infections produced by the 
intravenous inoculation of the same strain of M. tuberculosis, whereas 
Compound II is totally inactive against this infection. Absorption from 
the intestinal tract does not appear to be a factor, because Compound II 
is found in high levels in the circulatory system of orally medicated, M. 
tuberculosis infected mice. Compounds III and IV are also equally active 
against the TB organism in vitro (25 mcg./ml.), but against the systemic 
infection in mice, Compound III is four times more effective than Compound 
IV. Compound V is inactive against TB both in vitro and in vivo, although 
it is metabolized to Compound IV by the animals, as indicated by the 
appearance of Compound IV in the urine of mice that have been medicated 
orally with Compound V. 
Compounds III and IV are just as active in vitro against Staphylococcus 
aureus as they are against the M. tuberculosis organism. However, both 
compounds are completely inactive against systemic infections in mice 
produced by this same Staphylococcus aureus with equal medication regimens 
for both infections. It is an accepted fact that systemic infections 
caused by Staphylococcus aureus, because of the rapid development of high 
titers of circulating antistaphylococcal antibodies, are affected by 
humoral and not cellular immunity. 
(2) The dose-response curves for Compounds III and IV against systemic 
tuberculosis infections is bell-shaped, i.e. effectiveness of the 
compounds increases with increasing doses to an optimum dose level and 
then decreases with increasing dosage. Toxicity is not a factor in the 
latter decline in activity. This behavior indicates that the compounds do 
not act directly on the organism but rather act via some other biological 
system. On the other hand, isoniazid, which acts directly upon the 
invading organism, possesses a linear dose-response curve, which flattens 
out at the 100% effective dose and remains flat at increasing doses up to 
the toxic dose where mortality of the host occurs through drug toxicity. 
(3) The direct involvement of the thymus and stimulation of T-cells either 
in the thymus or in peripheral blood on oral administration of the 
compounds of the invention was shown by the following tests: 
(a) The thymi of mice medicated with the compounds of the invention are 
enlarged over either infected or non-infected, unmedicated controls, 
whereas the thymi of isoniazid-medicated animals are not substantially 
different from uninfected, unmedicated controls, thus indicating 
stimulation of the thymus by the compounds of the invention and the lack 
of such stimulation by isoniazid. 
(b) Depletion of T-cell population by the administration of anti-T-cell 
serum has the effect of cancelling out the effectiveness of the compounds 
of the invention against systemic tuberculosis infections. On the other 
hand, administration of anti-T-cell serum to isoniazid or rifamycin 
treated animals infected with tuberculosis, both of which drugs owe their 
anti-TB activity to their ability to attack the invading microorganism 
directly, produced almost no diminution in the effectiveness of the test 
compounds. 
(4) The activation of macrophages as one of the parameters in the immune 
response system, which are in turn modulated by the subject compounds, was 
shown by an experiment in which T-cell stimulation in tuberculosis 
infected animals by the subject compounds was markedly reduced by 
administration of anti-macrophage serum which substantially reduced the 
effectiveness of the compounds of the invention against systemic 
tuberculosis infections. On the other hand, anti-macrophage serum has 
virtually no effect on the effectiveness of isoniazid in the treatment of 
tuberculosis infected animals. 
(5) The anti-TB activities of the compounds of the invention are markedly 
reduced by the administration of trypan blue, a result which is believed 
due to inhibition of trypan blue of lysosomal enzymes produced by the 
macrophages [Beck et al., Science, 157: 1180-1182 (1967)]. On the other 
hand, the anti-TB activity of isoniazid is only moderately diminished by 
trypan blue. 
(6) Similarly the anti-TB activities of the compounds of the invention are 
blocked by simultaneous administration of dexamethasone, an adrenocortical 
steroid which is known to destroy leucocytes [Eisen, Immunology, Harper 
and Row Publishers, 2nd Ed., pages 473-474 (1974)]. 
(7) Finally, although the compounds of the invention possess no intrinsic 
anti-viral activity, yet they protect mice from challenge with Herpesvirus 
hominis. Certain of the compounds have furthermore been found to alleviate 
inflammatory arthritic conditions, to lower blood pressure and to lower 
blood glucose levels in animal models. The mechanisms that control 
recovery from Herpes infections require participation of cellular 
immunity, and as indicated above the test results against arthritic, 
hypertensive and hyperglycemic conditions are not inconsistent with the 
involvement of cell-mediated response. 
The compounds of the invention can be formulated for use by preparing a 
dilute solution in an organic medium in which the compounds are soluble, 
for example ethyl alcohol, or they can be formulated in conventional 
carriers, including sugars such as sucrose, lactose or maltose, for oral 
administration as tablets or capsules. 
BIOLOGICAL TEST RESULTS 
The general in vitro anti-bacterial activities of the compounds of the 
invention were determined using standard serial dilution procedures. 
Results so obtained are given in Table 1 below, the results being 
expressed in terms of the Minimum Inhibitory Concentration (MIC) in 
mcg./ml. The letters (a), (b), (c), (d), (e), (f), (g), (h) and (i) 
designate, respectively, the organisms S. aureus Smith, E. coli Vogel, E. 
coli 198, K. pneumoniae 39645, Pr. mirabilis MGH-1, Pr. vulgaris 9920, Ps. 
aeruginosa MGH-2, Strep. pyogenes C203 and M. tuberculosis H37Rv. 
Table 1 
__________________________________________________________________________ 
Compound No. 
(a) (b) (c) (d) (e) (f) (g) (h) (i) 
__________________________________________________________________________ 
I &gt;31.3 &gt;31.3 
&gt;31.3 &gt;31.3 &gt;31.3 &gt;31.3 &gt;31.3 
&gt;31.3 
3.1 
II &gt;125 &gt;62.5 
&gt;125 &gt;62.5 &gt;125 &gt;125 &gt;62.5 
&gt;62.5 
3.1 
III 31.3 &gt;62.5 
&gt;62.5 &gt;62.5 125 125 &gt;62.5 
31.3 6.25 
IV 31.3 &gt;62.5 
&gt;125 &gt;125 125 125 &gt;62.5 
31.3 6.25 
V &gt;125 &gt;62.5 
&gt;125 &gt;125 &gt;125 &gt;125 &gt;62.5 
&gt;62.5 
3.1 
__________________________________________________________________________ 
The effectiveness of the compounds of the invention against systemic 
Mycobacterium tuberculosis infections when administered orally to mice was 
shown by a test procedure in which mice were infected by intravenous 
innoculation of 0.1 cc. of a 2 mg. (moist weight)/cc. suspension of M. 
tuberculosis, strain H37Rv grown in Youman's medium for two weeks at 
37.5.degree. C. The test animals were then treated daily with graded oral 
doses of the test compound for four weeks, and all mice, regardless of 
whether they died of the infection during the experiment or were 
sacrificed at the termination of the experiment, were autopsied at the 
time of death. At autopsy, the TB lung lesions were scored and given one 
of the following numerical values: 
0=(normal lung) 
15=.+-. (lung congestion and enlargement-no lesions) 
33=+ (lung congestion and enlargement-few small lesions) 
66=++ (TB lesions cover 50% of the lungs) 
100=+++ (TB lesions cover 100% of the lungs). 
A drug regimen that produces a survival rate of 90% to 100% and an average 
lung score of 25 or lower is considered to be outstandingly active. The 
results are given in Table 2 along with data for isoniazid, which is 
included for purposes of comparison. The data are given in each case in 
terms of the % survival (hereinafter % S), the lung score (hereinafter 
L.S.) and also in terms of the ratio of the lung score of control animals 
to the lung score of the test animals (hereinafter the C/T ratio). The 
higher this ratio, the more effective the test compound is considered to 
be. 
Table 2 
______________________________________ 
Med- 
ica- Dose in mg./kg./day 
tion 0 5 3.1 6.25 12.5 25 50 
______________________________________ 
In- 
fec- 
ted 
Con- 0% 
trol (96.6) 
Ison- 100% 
iazid (16.8) 
5.75 
Cpd. 20% 40% 100% 100% 100% 
I (84.8) 
(55.8) 
(28.8) 
(18.6) 
(21.9) 
1.14 1.73 3.35 5.19 4.10 
Cpd. 20% 0% 40% 40% 
II (89.9) 
(81.5) 
(73.2) 
(73.2) 
1.07 1.19 1.32 1.32 
Cpd. 30% 10% 40% 50% 60% 
III (71.3) 
(83.1) 
(34.2) 
(40.8) 
(32.4) 
1.35 1.16 2.82 2.38 2.98 
Cpd. 30% 80% 90% 70% 0% 
IV (60.9) 
(27.3) 
(22.2) 
(27.3) 
(52.8) 
1.59 3.54 4.35 3.54 1.83 
Cpd. 10% 
V (93.3) 
1.04 
______________________________________ 
Using the same test procedure as that described above in connection with 
the data presented in Table 2, the fact that the compounds of the 
invention are active in vivo against TB infections in mice only when 
administered orally was shown by the data presented in Table 3 below where 
the routes of administration (oral, subcutaneous and intraperitoneal) are 
indicated by the respective designations p.o., s.c. and i.p., and where as 
before %S and L.S. represent percent survival and lung score, 
respectively, and C/T is the ratio of the lung score of control and test 
animals. 
Table 3 
__________________________________________________________________________ 
P.O. S.C. I.P. 
Cpd. 
Dose 
% S L.S. 
C/T 
% S 
L.S. 
C/T 
% S 
L.S. 
C/T 
__________________________________________________________________________ 
I 3.125 
50 37.5 
2.58 
30 71.3 
1.35 
20 59.7 
1.62 
I 6.25 
60 33.9 
2.85 
30 89.9 
1.07 
0 66.4 
1.45 
I 12.5 
100 22.2 
4.35 
10 66.0 
1.46 
0 54.2 
1.78 
I 25 100 18.6 
5.19 
10 84.7 
1.14 
10 54.3 
1.78 
I 50 100 16.8 
5.75 
20 66.1 
1.46 
50 40.7 
2.37 
III 3.125 
30 79.6 
1.26 
30 73.1 
1.37 
60 32.5 
3.1 
III 6.25 
80 30.6 
3.27 
50 59.3 
1.69 
60 47.5 
2.1 
III 12.5 
70 37.5 
2.80 
40 62.6 
1.60 
50 49.5 
2.01 
III 25 70 34.0 
2.94 
10 86.5 
1.16 
0 40.8 
2.45* 
III 50 100 25.2 
3.97 
10 86.5 
1.16 
0 30.6 
3.27* 
__________________________________________________________________________ 
*Toxic levels i.p. mice died before lesions could develop. 
Some data obtained in tests on the compounds of the invention indicate, but 
fail to completely establish, that the compounds are metabolized in the 
animal body. Thus in urinary antisepsis tests, the compounds of the 
invention were administered orally to groups of three normal rats at a 
dose level of 100 mg./kg. Half of the dose was given in the morning and 
the other half eight hours later. The rats were placed in metabolism cages 
after the first medication, and the pooled urine from the three rats were 
collected for twenty-four hours after the first medication. The urine was 
sterilized by membrane filtration and tested for antibacterial activity 
against standard screening organisms. The Maximum Inhibitory Dilution 
(MID) of the urine against each organism is given in Table 4 where as 
before the organisms S. aureus Smith, E. coli Vogel, E. coli 198, K. 
pneumoniae 39645, Pr. mirabilis MGH-1, Pr. vulgaris 9920 and Ps. 
aeruginosa MGH-2 are identified by the letters (a), (b), (c), (d), (e), 
(f) and (g), respectively. 
Table 4 
______________________________________ 
Organism 
Cpd. I Cpd. II Cpd. III 
Cpd. IV Cpd. V 
______________________________________ 
(a) 1:8 1:8 1:32 1:64 1:32 
(b) &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 
(c) 1:32 1:128 
(d) &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 
(e) 1:16 1:8 1:4 1:16 1:32 
(f) &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 
(g) &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 &lt;1:2 
______________________________________ 
Comparison of these data with that shown in Table 1 shows that in the 
urinary antisepsis test, certain of the compounds, especially Compounds I, 
II and V display an entirely different profile of in vitro activity 
against the same series of microorganisms than found in the earlier tests, 
thus indicating that metabolites in the urine are involved. 
A further indication that a metabolite of Compounds I, III and IV may in 
some way be involved in the immune response mechanism is provided by data 
obtained showing that the anti-TB activity of the compounds can be blocked 
by intraperitoneal injection of 30 mg./kg./dose two times a day of SKF 
525A [2-diethylaminoethyl-.alpha.,.alpha.-diphenylvalerate hydrochloride], 
a compound which interferes with the metabolism of other administered 
compounds by inhibition of liver microsomal enzymes [Rogers et al., J. 
Pharmacol. Exptl. Therap. 146, 286-293 (1964)]. Data so obtained which 
show the blocking effect of SKF 525A on the anti-TB activity of each of 
Compounds I, III and IV, but not that of isoniazid, which does not act by 
cell-mediated immunity, are given in Table 5 where as before %S and L.S. 
represent % survival and lung score, respectively. 
Table 5 
__________________________________________________________________________ 
Control Cpd. I* 
Cpd. III* 
Cpd. IV* 
Isoniazid* 
% S L.S. 
% S L.S. 
% S L.S. 
% S 
L.S. 
% S L.S. 
__________________________________________________________________________ 
(A) 
5 96.6 
100 16.8 
100 18.6 
60 46 100 15.0 
(B) 
10 96.6 
40 66.3 
50 69.7 
20 79.7 
90 27.3 
__________________________________________________________________________ 
(A) Without SKF 525A 
(B) With SKF 525A 30 mg./kg./dose B.I.D. 
*12.5 mg./kg./day divided 
Using a high pressure liquid chromatographic assay method sensitive to 
0.007 mcg./ml. no levels of Compound I could be detected in the blood 
plasma of mice that had been orally medicated daily for four weeks with 
dose levels of 3.1, 6.25, 12.5 and 25 mg./kg./day of the compound, doses 
that were found to be highly effective against tuberculosis infections. 
These results demonstrate that the alleviation of the tuberculosis disease 
condition is not attributable to Compound I as such. 
The structures of any metabolites that may be produced from any of the 
subject compounds have not been determined, and in fact it is not known 
whether the immune stimulation produced by the compounds is due to the 
structures themselves or to some metabolite. 
Further evidence for the oral activity of Compounds I, III and IV against 
M. tuberculosis infections was obtained by administration of the compound 
to guinea pigs infected with M. tuberculosis H37Rv using the same 
procedure as described above in connection with the data presented in 
Table 2 recording results obtained in mice. However, since TB infections 
in guinea pigs produce lesions which, rather than being confined to the 
lungs as in mice, are produced throughout the body, especially in the 
spleen, the liver, the lungs and the lymph nodes, a different scoring 
system for each of these organs is used based on the same scale as used in 
the mouse model described above, and a total score for the test animals is 
calculated as the sum of the individual organ scores. Data so obtained on 
Compounds I and III and for isoniazid (designated "Ref.") are given in 
Table 6. As before doses are expressed in mg./kg., and %S represents 
percent survivors. 
Table 6 
______________________________________ 
Organ Score 
Lymph 
Cpd. Dose % S Spleen 
Liver Lung Node Total 
______________________________________ 
Control 
-- 44.6 20.7 14.3 15.0 11.7 61.7 
I 6.25 80 12 8 10 5 35 
I 12.5 80 8 6 6 6 26 
I 25 100 8 9 5 3 25 
I 50 60 0 0 2 2 4 
III 6.25 80 12 11 7 6 36 
III 12.5 100 26 17 10 9 62 
III 25 80 11 9 6 7 33 
III 50 60 9 11 6 7 33 
Ref. 1.5 100 5 5 4 7 13 
Ref. 3.1 100 2 3 6 2 13 
Ref. 6.25 80 1 1 3 0 5 
Ref. 12.5 80 0 0 2 0 2 
______________________________________ 
Although two of the compounds of the invention (III and IV) have been shown 
to be as active in vitro against Staphylococcus aureus as they are against 
the TB organism, they are inactive on in vivo administration against 
staphylococcus infections in mice, a further indication that the compounds 
do not either act as such directly on the organism or do not activate the 
humoral immune system and thus that they likely act by stimulation of the 
cell-mediated immune system. Data so obtained, expressed in terms of 
number of survivors out of a total test group at given dose levels, are 
given in Table 7 below. 
Table 7 
______________________________________ 
Dose 
Cpd. mg/kg./day M. tuberculosis 
S. aureus 
______________________________________ 
III 3.1 0/10 
III 6.25 6/10 
III 12.5 10/10 
III 25 10/10 
III 50 10/10 0/10 
III 100 0/10 
III 200 0/10 
IV 3.1 0/10 
IV 6.25 3/10 
IV 12.5 10/10 
IV 25 10/10 
IV 50 10/10 1/10 
IV 100 0/10 
IV 200 0/10 
______________________________________ 
Stimulation of the thymus by one of the subject compounds (Compound I) when 
administered orally to non-infected mice in comparison with either TB 
infected or non-infected control mice or in comparison with isoniazid 
medicated non-infected controls is shown by the data given in Table 8. 
Compound I and isoniazid were administered at respective oral doses of 
12.5 and 5 mg./kg./day for four weeks, and all animals were autopsied at 
the same age. As shown by the data, the thymi of mice medicated with 
Compound I are greatly enlarged in comparison with the thymi of either 
non-medicated infected or uninfected mice or when compared with 
non-infected isoniazid-medicated mice. 
Table 8 
______________________________________ 
TB Thymus 
Cpd. Infection Ave. Wt. (mg.) 
______________________________________ 
None None 57.4 
I None 71.1 
Isoniazid None 57.2 
None Yes 38.6 
______________________________________ 
Further evidence of the involvement of the thymus and of T-cell stimulation 
in the mechanism of action of the subject compounds in alleviating 
tuberculosis infections is shown by the data in Table 9 which shows the 
effect of the administration of anti-T-cell serum. The latter is an immune 
serum prepared by immunizing rabbits with T-cells isolated from the same 
strain of mice used in the TB studies. This hyperimmune serum, when 
injected back into mice, severely depletes the T-cell population. In this 
study, each of four groups of twenty tuberculosis infected mice per group 
were randomly divided in half. One half of each group received 0.25 ml. of 
anti-T-cell immune serum twice weekly for four weeks subcutaneously in 
addition to the anti-tuberculosis medication, and the other half received 
only the anti-tuberculosis medication. In Group I, none of the mice 
received anti-TB medication and half received anti-T-cell serum. In Groups 
2, 3 and 4 all mice in each group received an oral medication of 12.5, 25 
and 25 mg./kg./day of Compounds I, III and IV, respectively, and half of 
each of these groups received anti-T-cell serum. In Group 5 all of the 
mice received oral medication of isoniazid at 6.25 mg./kg./day and half 
received anti-T-cell serum. In Group 6, all of the mice received oral 
medication of rifamycin at 12.5 mg./kg./day, and half of the group 
received anti-T-cell serum. Compound I, when administered at 12.5 
mg./kg./day for a four week period, produced 100% survival and a lung 
score of 15 (no visible TB lesions), but when anti-T-cell serum was 
administered to the other half of this group, the survival rate dropped to 
20%, and the lung score soared to 89.9. Similarly, Compound III 
administered at 25 mg./kg./day for four weeks produced 80% survival and a 
lung score of 40.4, but administration of anti-T-cell serum to the other 
half caused the survival rate to drop to 30% and the lung score to more 
than double to 86.6. Compound IV administered at 25 mg./kg./day for four 
weeks produced a 60% survival rate and a lung score of 54.3, while 
administration of anti-T-cell serum to the other half of this group caused 
the survival rate to drop to 20% and the lung score to almost double to 
83.3. These data thus clearly demonstrate that the anti-tuberculosis 
activities of Compounds I, III and IV is virtually eliminated by the 
simultaneous administration of anti-T-cell immune serum. In contrast, 
neither isoniazid nor rifamycin, two well known anti-TB drugs which 
directly inhibit the organism, lose activity on simultaneous 
administration of anti-T-cell serum to the infected animals. The data so 
obtained are given in Table 9 below. 
Table 9 
______________________________________ 
Drug Anti-T-Cell Thymus 
Regimen Serum % S L.S. Ave. Wt. (mg.) 
______________________________________ 
Infected 
None 0 100 38.6 
Control 2 .times. weekly 
0 96.6 12.4 
Cpd. I None 100 15 128.8 
2 .times. weekly 
20 89.9 17.9 
Cpd. III 
None 80 40.4 96.4 
2 .times. weekly 
30 86.6 18.3 
Cpd. IV None 60 54.3 85.4 
2 .times. weekly 
20 83.3 18.7 
Isoniazid 
None 100 15 117.6 
2 .times. weekly 
100 20.4 89.7 
Rifamycin 
None 90 18.6 98.2 
2 .times. weekly 
90 30.3 64.8 
______________________________________ 
Evidence that the subject compounds are involved in the stimulation of 
macrophages through the immune system is provided by the data presented in 
Table 10 which shows the inhibitory effect on the anti-TB activity of the 
compounds of the invention by the administration of anti-macrophage serum. 
Antimacrophage serum (AMS) was administered at 0.15 ml./dose (i.p.) at two 
and one days prior to TB infection and at two, five and eight days post 
infection. The procedure used is similar to that used in the anti-T-cell 
serum test described above. 
Table 10 
______________________________________ 
Medication Dose Ave. Days Surv. 
%S L.S. C/T 
______________________________________ 
Infected Controls 
-- 24.3 40 80 1.04 
AMS Infected 
-- 24.3 40 83.1 -- 
Controls 
AMS + Cpd. I 
12.5 25.1 50 58 1.45 
AMS + Cpd. III 
2.5 25 50 61 1.36 
AMS + Isoniazid 
6.25 31 100 15 5.54 
______________________________________ 
As shown by the data, neither Compound I nor Compound III produce any 
improvement in the survival rate of TB infected mice treated with AMS at 
doses previously found to be effective against this infection. In 
contrast, the effectiveness of isoniazid against TB infection is in no way 
diminished by administration of AMS. 
Evidence of the involvement of macrophages in the anti-TB activity of one 
of the subject compounds is also shown by experiments in which 
administration of trypan blue, a macrophage toxin, markedly reduces the 
anti-TB activity of Compound I in tuberculosis infected mice. In contrast, 
the activity of isoniazid, whose anti-TB activity does not depend on 
involvement of macrophages and the immune system, is only moderately 
affected by trypan blue administration. Data so obtained are given in 
Table 11. The trypan blue was administered at a dose of 4 mg. two times a 
week intraperitoneally, while the reference drug, isoniazid, was 
administered at 5 mg./kg./day for four weeks. As before %S, L.S. and C/T 
represent percent survivors, lung score and the ratio of the lung score in 
control to test animals, respectively. Administration of trypan blue is 
indicated in the column headed Tr. Bl. 
Table 11 
______________________________________ 
Dose 
Medication 
(mg./kg.) Tr. Bl. % S L.S. C/T 
______________________________________ 
Infect. Controls 
-- No 0 100 -- 
Isoniazid 5 No 100 15 6.67 
Trypan Blue 
-- Yes 10 93.2 1.07 
Isoniazid 5 Yes 90 33.9 2.95 
Cpd. I 6.25 No 70 47.4 2.11 
Cpd. I 12.5 No 100 16.8 5.95 
Cpd. I 25 No 100 16.8 5.95 
Cpd. I 6.25 Yes 40 73 1.28 
Cpd. I 12.5 Yes 70 76 1.22 
Cpd. I 25 Yes 80 52.8 1.77 
______________________________________ 
As can be seen, the trypan blue had only moderate effect on either the 
survival rate or the lung scores in animals administered isoniazid, while 
administration of trypan blue with Compound I dramatically reduced the 
effectiveness of the latter. 
Further studies have shown that trypan blue, either alone or in combination 
with Compound I, enhances phagocytic activity. This is demonstrated by 
measuring the rate of clearance of radio labelled colloid from test 
animals administered either trypan blue, Compound I alone or trypan blue 
and Compound I together and comparing the results with those obtained with 
unmedicated controls. The procedure used is described as follows: 
Sprague-Dawley rats, weighing 200 g. were lightly anesthetized with 
pentobarbital, and a gelatinized lipid emulsion with a .sup.131 I label 
was injected at a dose of 50 mg./kg. via tail vein and blood samples taken 
at two minute intervals for the first ten minutes. After a lapse of 
fifteen minutes from the time of administration of the radio labelled 
emulsion, the animals were killed, and the liver, lungs and spleen 
removed, weighed and tissue samples of each taken for determination of the 
amount of colloid present. A Baird-Atomic gamma counter was employed to 
assess radioactivity of the blood and tissue samples so that blood 
clearance rates and tissue deposition on a per gram and total organ basis 
could be determined. As shown by the data in Table 12 below, Compound I at 
either 3, 20 or 50 mg./kg./day (p.o.) enhanced clearance rates of the 
colloid. However trypan blue, either alone or in combination with Compound 
I, accelerated the rate of clearance even more, thus indicating enhanced 
phagocytic activity by the combination of Compound I and trypan blue 
(T.B.). 
Table 12 
______________________________________ 
Clearance % Injected Drug In: 
Medication Days (t1/2) Liver Lung Spleen 
______________________________________ 
Control 10.27 49.09 0.89 5.40 
Trypan Blue 
7 8.94 44.79 1.81 3.06 
Trypan Blue 
14 7.08 71.14 1.99 4.51 
Trypan Blue 
20 9.46 76.00 0.78 4.23 
Trypan Blue 
28 8.81 63.40 0.84 6.38 
Cpd. I 8 7.26 58.29 0.77 4.36 
Cpd. I 15 10.35 55.17 1.10 6.49 
Cpd. I 22 8.59 60.10 0.84 5.83 
Cpd. I 29 9.68 52.27 0.92 4.63 
Cpd. I + T.B. 
7 6.83 75.06 1.89 1.65 
Cpd. I + T.B. 
14 7.02 70.03 0.85 4.48 
Cpd. I + T.B. 
21 8.13 55.86 0.68 3.90 
Cpd. I + T.B. 
28 7.83 59.96 0.82 5.58 
______________________________________ 
Reduction in the number of leucocytes, including macrophages and 
T-lymphocytes, in M. tuberculosis infected mice medicated with the subject 
compounds was also shown by studies in which subcutaneous administration 
of either 1 mg./kg./day or 10 mg./kg./day of dexamethasome (Dexa.) was 
shown to sharply inhibit the anti-TB activity of Compound I in comparison 
with either non-dexamethasone medicated controls or isoniazid-medicated 
animals. The data so obtained are given in Table 13. 
Table 13 
______________________________________ 
Dose 
Medication Dose Dexa. % S L.S. C/T 
______________________________________ 
Infected Cont. 
-- -- 5 100 -- 
Isoniazid 5 -- 100 15.0 6.66 
Cpd. I 6.25 -- 60 50.8 1.97 
Cpd. I 12.5 -- 100 18.6 5.38 
Cpd. I 25 -- 90 18.6 5.38 
Dexa. -- 1 0 100 1.0 
Isoniazid 5 1 100 15.0 6.66 
Cpd. I 6.25 1 10 96.6 1.03 
Cpd. I 12.5 1 30 93.2 1.07 
Cpd. I 25 1 100 40.8 2.45 
Dexa. -- 10 10 100 1.0 
Isoniazid 5 10 90 16.8 5.95 
Cpd. I 6.25 10 40 78.8 1.28 
Cpd. I 12.5 10 0 83.0 1.20 
Cpd. I 25 10 10 83.0 1.20 
______________________________________ 
The effectiveness of Compound I in the treatment of virus infections known 
to be susceptible to treatment by cell-mediated stimulation of the immune 
response system was shown by in vivo tests against Herpesvirus 
hominis-Type 2 although the drug has no direct effect on the same virus 
when tested in tissue culture medium. Thus two experiments were conducted 
in which mice innoculated intravaginally with Herpesvirus hominis-Type 2 
received Compound I orally for two weeks prior to infection and continuing 
for two weeks post infection. The disease produced by this innoculation is 
evidenced by herpetic vesicles and inflammation of the vulva which appear 
about four days after innoculation. This is followed in two or three days 
by hind leg paralysis and finally death. In the first study carried out in 
mature mice, the infection which produced 50% mortality in the untreated 
controls, was lighter and more easily controlled by the drug than in the 
second study carried out in young mice. Thus the mice were somewhat 
younger and thus more susceptible to the disease in the second study. Data 
so obtained are given in Table 14 in terms of percent survival. From the 
data it will be seen that at dose levels of 6.25 to 12.5 mg./kg., dramatic 
improvements over controls were obtained in both studies. 
Table 14 
______________________________________ 
Study Dose (mg./kg.) Cpd. I 
No. Control 3.1 6.25 12.5 25 50 100 200 
______________________________________ 
1 50 70 80 100 100 -- -- -- 
2 15 15 70 60 50 60 40 30 
______________________________________ 
The effectiveness of the subject compounds in the treatment of inflammatory 
conditions was demonstrated by results obtained in anti-inflammatory 
activity tests on oral administration in mice using the 
carrageenin-induced foot edema test method described by Van Arman et al., 
J. Pharmacol. Exptl. Therap. 150, 328 (1965), a modification of the 
procedure described by Winter et al., Proc. Soc. Exp. Biol. Med. 111, 544 
(1962), and the adjuvant-induced arthritis test described by Pierson, J. 
Chronic Diseases 16, 863 (1963) and Glenn et al., Am. J. Vet. Res. 26, 
1180 (1965). The results so obtained are given in Table 15. Doses are 
expressed in terms of millimoles per kg., and the results are expressed in 
terms of percent inhibition of the inflammatory condition. The 
designations A.A. and C.E. represent the adjuvant-induced arthritis and 
carrageenin-induced edema tests, respectively. 
Table 15 
______________________________________ 
Cpd. Dose (mM/kg.) A.A. C.E. 
______________________________________ 
I 0.023 33* -- 
0.07 50** -- 
0.08 -- 13 
0.21 69** -- 
0.324 -- 22 
II 0.023 32** -- 
0.07 76** -- 
0.21 79** -- 
III 0.023 27 -- 
0.07 28* -- 
0.21 31* -- 
IV 0.023 28* -- 
0.07 21 -- 
0.21 15 -- 
V 0.023 23 -- 
0.07 25 -- 
0.21 58** -- 
______________________________________ 
*Statistically significant from positive controls p.ltoreq..05 
*Statistically significant from positive controls p.ltoreq..01 
Two of the subject compounds (Compounds I and II) were found to be active 
as antihypertensive agents on oral administration in spontaneously 
hypertensive rats. The spontaneously hypertensive rat (SHR) is a strain of 
genetically hypertensive animals developed from Wistar rats by Okamoto et 
al., Jap. Circ. J., 27, 282-293 (1963) after selective inbreeding. Unlike 
any other hypertensive animal model, the SHR requires no surgical 
intervention, and it is widely recognized as the closest model to 
essential hypertension in man. Furthermore, the hypertension in these rats 
is not due to any of the known causes of secondary hypertension, and the 
blood pressure increases progressively with age. Complications often 
observed in human essential hypertension such as cardiac, renal and 
vascular changes are also observed in the SHR. The procedure used is 
described as follows: Each compound to be screened is tested in five SHR's 
whose base line blood pressure has been determined earlier in the day. The 
test compounds are given orally, suspended in 1% gum tragacanth in a total 
volume of 1 ml./kg. Systolic blood pressure measurements at 2, 6 and 24 
hours after administration of the test compound are obtained with a 
photoelectric tensometer (ankle rubber cuff method) as described by 
kersten et al., J. Lab. Clin. Med. 32, 1090-1098 (1947). Compound I 
suspended in gum tragacanth was administered at daily oral doses of 50 
mg./kg. for twelve consecutive days, and Compound II suspended in gum 
tragacanth (GT) was administered at 50 mg./kg. for seven days followed by 
a dose of 100 mg./kg. for the next five days. A control group run side by 
side in each study received only the gum tragacanth. Data so obtained are 
given in Table 16 below, results being recorded in terms of the mean blood 
pressure and the scientific error for each reading. 
3 Table 16 
Pre-Med 1 2 
3 4 5 7+ 8 9 10 11 12 Medication Baseline B.P. 2 6 24 2 6 24 2 6 24 2 6 2 
4 2 6 24 2 6 24 6 24 2 6 24 6 24 2 6 
1%GT 182 178 178 180 183 184 180 184 189 193 -- 188 189 188 184 193 
194 193 186 184 191 188 183 192 189 195 195 197 9.1 5.2 5.8 7.8 5.8 7.0 
8.4 4.5 7.6 10. -- 7.9 11.7 9.8 11.2 9.1 7.2 8.4 9.4 5.3 7.2 5.6 7.3 
13.1 6.7 7.5 5.0 6.6 ** * * ** ** ** ** ** ** ** ** ** ** 
** ** Cpd 183 182 182 183 170 186 183 155 163 165 -- 158 166 165 163 172 
152 162 155 142 151 148 151 147 149 140 145 143 I 7.2 5.6 7.0 6.4 6.6 
6.9 5.0 6.0 5.7 9.4 -- 10.2 6.1 5.3 4.2 6.3 7.9 6.6 8.1 7.1 6.5 5.6 5.2 
7.4 3.8 7.4 6.2 4.2 1%GT 183 178 182 174 178 182 176 186 181 176 191 182 
173 182 186 187 182 182 -- -- 192 188 188 -- -- 188 187 180 6.2 4.6 4.5 
4.8 6.7 5.1 4.1 8.7 3.9 8.7 5.4 9.2 6.7 5.6 8.7 3.2 6.1 8.8 -- -- 6.4 
5.6 5.6 -- -- 2.2 4.8 4.7 * ** * ** ** * * ** ** ** 
II 6.2 2.7 2.7 3.7 3.2 5.5 5.4 8.1 6.0 6.7 2.1 5.9 4.6 6.2 5.2 1.8 4.4 
5.4 -- -- 7.4 5.7 3.6 -- -- 2.8 3.0 3.0 
+Medication on weekend (Days 6 and 7) but no blood pressure measurements 
taken 
*Significantly different from corresponding control p&lt;0.05 
**Significantly different from corresponding control p&lt;0.01 
Compound I was also found active in the adrenal-regeneration hypertensive 
rat test [Skelton, Proc. Soc. Exp. Biol. Med. 90, 342 (1955)]. As in the 
SHR test, the compound was administered at single daily doses of 50 
mg./kg./day for twelve consecutive days in a gum tragacanth suspension. A 
control group received only the gum tragacanth. Data so obtained expressed 
in terms of the mean blood pressure and the scientific error for each 
reading are given in Table 17 below. 
3 Table 17 
Pre- Med Base- Medi- line 1 2 3 4 5 7+ 8 9 10 11 12 
cation B.P. 2 6 24 2 6 24 2 6 24 2 6 24 2 6 24 2 6 24 2 6 24 2 6 24 2 6 
24 2 6 
1%GT 194 189 191 196 191 192 194 192 184 198 196 191 195 194 195 197 
192 192 196 191 192 195 194 196 194 187 189 193 194 194 10.2 11.7 9.5 
12.0 8.8 10.7 12.3 14 17.7 8.4 11 9.7 12.7 13 12.5 13.0 12.5 12.6 11.2 
13.2 9.4 11.9 12.7 12.9 11.5 13.4 13.8 11 10.8 10.2 * * ** 
* * * ** ** * Cpd I 194 190 186 195 187 186 184 177 166 152 158 
158 161 164 159 148 165 166 170 167 171 161 153 151 153 164 161 140 151 
148 7.9 14.6 7.9 7.8 10.8 11 9.8 9.0 6.2 16.4 13 10.2 9.7 8.9 11.1 5.4 
9.3 6.9 6.4 14.9 9.5 8.7 13.6 11.2 10.5 8.1 6.9 6.5 6.4 10.9 
*Medication on weekend (Days 6 and 7) but no blood pressure measurements 
taken 
*Significantly different from corresponding control p&lt;0.05 
*Significantly different from corresponding control p&lt;0.01 
The data presented in Tables 16 and 17 show that a 50 mg./kg. single daily 
oral dose of Compound I resulted in an anti-hypertensive effect that 
became apparent on the third day, and reductions in blood pressure on the 
order of 40 to 50 mm. Hg had been reached. Compound II in the same test 
showed somewhat the same pattern of blood pressure lowering, but the 
magnitude of the lowering at the end of the test period was somewhat less 
(i.e. 20-30 mm. Hg). In the adrenal regeneration model, Compound I at 
daily oral doses of 50 mg./kg. for twelve consecutive days produced an 
anti-hypertensive response beginning on the third day and reached a 
lowering of 40-50 mm. Hg below base line at the end of the study. 
Certain compounds of the invention have also been found to have 
hypoglycemic activity in alloxan and streptozotocin-induced diabetes in 
rats. In the test procedures used, diabetes was produced in male, 
Sprague-Dawley rats weighing approximately 150 g. by the administration of 
either alloxan monohydrate, 160 mg./kg. (s.c.), or streptozotocin, 65 
mg./kg. (i.v.), after an overnight fast. Fasting blood glucose levels were 
obtained once each week for three weeks using a modification of the 
procedure described by Hoffman, J. Biol. Chem. 120, 51(1937) which 
involves the measurement of the reduction of yellow ferricyanide ion to 
colorless ferrocyanide by glucose. Animals having fasting blood glucose 
levels of &lt;250 mg./dl. for the last two weeks were distributed into groups 
such that all groups in a test had a similar distribution and mean fasting 
blood glucose level. Normal rats were similarly distributed on the basis 
of a single fasting blood glucose level. The test compounds were suspended 
in 1% gum tragacanth and administered orally in 5 ml./kg. twice a day. One 
group in each test was given 1% gum tragacanth as a control, and fasting 
blood glucose levels were determined for each group at weekly intervals. 
Results, expressed in terms of mg. of blood glucose/dl., obtained in 
streptozotocin-treated animals are given in Table 18, and results obtained 
in the alloxan model are given in Table 19. In the streptozotocin model, 
results are the mean.+-.s.e. of nine rats per group, while in the alloxan 
model, results are the mean.+-.s.e. of thirteen rats per group. 
Table 18 
__________________________________________________________________________ 
Week 1 2 3 
Medication 
Dose 
Post Med. Hr. 
0 3 6 0 3 6 0 3 6 
__________________________________________________________________________ 
1% G.T. 
-- 358.+-.15 
377.+-.18 
351.+-.26 
360.+-.13 
376.+-.11 
358.+-.13 
367.+-.10 
405.+-.8 
375.+-.11 
Cpd. I. 12.5 
335.+-.13 
342.+-.16 
339.+-.14 
326.+-.19 
350.+-.20 
346.+-.22 
327.+-.15 
366.+-.15 
358.+-.12 
Cpd. I 
50 284.+-.28 
264.+-.35 
244.+-.34 
251.+-.26 
237.+-.27 
206.+-.32 
252.+-.19 
224.+-.26 
169.+-.27 
%.increment. vs. (-30%) 
(-37%) 
(-42%) 
(-31%) 
(-45%) 
(-55%) 
Control 
1% G.T. 
-- 357.+-.11 
378.+-.9 
378.+-.5 
358.+-.12 
366.+-.5 
353.+-.12 
354.+-.11 
386.+-.6 
378.+-.5 
Cpd. V 
12.5 344.+-.10 
356.+-.17 
336.+-.24 
345.+-.7 
344.+-.9 
311.+-.22 
335.+-.6 
337.+-.11 
309.+-.26 
Cpd. V 
25 323.+-.11 
294.+-.20* 
252.+-.34** 
278.+-. 24* 
237.+-.33* 
196.+-.37* 
292.+-.27 
254.+-.37* 
216.+-.45* 
%.increment. vs. 
Control 
Cpd. (-22%) 
(-33%) 
(-22%) 
(-35%) 
(-44%) 
(-18%) 
(-34%) 
(-43%) 
Cpd. V 
50 335.+-.8 
282.+-.17** 
236.+-.30** 
278.+-.29 
202.+-.37** 
217.+-.41* 
203+33** 
147.+-.22** 
121.+-.24** 
%.increment. vs. (-25%) 
(-38%) (-45%) 
(-39%) 
(-43%) 
(-62%) 
(-68%) 
Control 
__________________________________________________________________________ 
*Significantly different from mean of vehicle control group p&lt;0.01 
**Signficantly different from mean of vehicle control group p&lt;0.001 
Table 19 
__________________________________________________________________________ 
Week 1 2 3 
Medication 
Dose 
Post Med. Hr. 
0 3 6 0 3 6 0 3 6 
__________________________________________________________________________ 
1% G.T. 325.+-.30 
348.+-.32 
322.+-.31 
352.+-.11 
380.+-.11 
383.+-.9 
352.+-.17 
388.+-.14 
376.+-.12 
Cpd. V 
50 299.+-.32 
263.+-.32 
277.+-.31 
220.+-.32* 
165.+-.27** 
119.+-.14** 
185.+-.34** 
148.+-.38 
139.+-.39 
%.increment. vs. (-38%) 
(-57%) 
(-69%) 
(-47%) 
(-62%) 
(-63%) 
Control 
__________________________________________________________________________ 
*Significantly different from mean of vehicle control group p&lt;0.01 
**Significantly different from mean of vehicle control group p&lt;0.001