Method and composition for treating HIV-type 1 infections

A method of treating mammals infected with HIV-1, or AIDS by administering a therapeutically effective amount of a compound represented by formula I ##STR1## or a pharmaceutically acceptable salt or ester thereof or stereoisomer thereof and pharmaceutical compositions containing it are disclosed.

BACKGROUND OF THE INVENTION 
This invention is directed to a method of treating a mammal infected with 
human immunodeficiency virus type 1, which comprises administering to such 
mammals a therapeutically effective amount of a dipeptide compound 
represented by the formula I 
##STR2## 
or a pharmaceutically acceptable salt or ester thereof or a stereochemical 
isomer thereof. 
The dipeptide compound of formula I is disclosed by R. Cooper et al. in The 
Journal of Antibiotics, 1988, Vol. 41 (No. 1 ), pp 13-19, to be an 
antifungal compound which was produced by fermentation of the 
microorganism Micromonospora sp. 
The human immunodeficiency virus type 1 ("HIV-1") is now generally 
considered to be the, cause of acquired immunodeficiency syndrome 
("AIDS"). See the editorial by D. Baltimore and M. B. Feinberg in The New 
England Journal of Medicine, 1989, Vol. 321 (No. 24), pp 1673-1675. The 
human immunodeficiency virus type 2 ("HIV-2") is a cause of AIDS in West 
Africa. 
People infected with HIV-1, HIV-2, AIDS and AIDS-Related Complex ("ARC") 
are being treated with various anti-HIV agents such as zidovudine (AZT), 
2'-3'-dideoxycytidine (DDC), 2',3'-dideoxyinosine (DDI) and certain 
tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and-thione 
("TIBO") derivatives. However, the AZT, DDC and DDI anti-HIV agents are 
relative highly cytotoxic to human lymphocytes uninfected by HIV-1 (i.e., 
have a relatively low margin of safety). The synthesis of the TIBO 
compounds are complex, time-consuming and provide only small amounts of 
these compounds. 
There is still a need for an anti-HIV-1 agent which is not highly cytotoxic 
and can be synthesized simply and in high yield. 
SUMMARY OF THE INVENTION 
The present invention provides a method of treating a mammal infected with 
human immunodeficiency virus type 1 which comprises administering to said 
mammal a therapeutically effective amount of a compound represented by the 
formula I: 
##STR3## 
or a pharmaceutically acceptable salt or ester thereof or a stereochemical 
isomer thereof. 
The present invention further provides a method of treating a mammal 
infected with acquired immune deficiency syndrome caused by human 
immunodeficiency virus type 1 which comprises administering to said mammal 
a therapeutically effective amount of a compound represented by the 
formula I 
##STR4## 
or a pharmaceutically acceptable salt or ester thereof or a stereochemical 
isomer thereof. 
The present invention also provides a pharmaceutical composition for 
treating a mammal infected with human immunodeficiency virus type 1, 
acquired immunodeficiency syndrome or acquired immunodeficiency 
syndrome-related complex comprising a therapeutically effective amount of 
a compound represented by formula I, together with a pharmaceutically 
acceptabler carrier therefor wherein the human immunodeficiency virus is 
implicated as the cause of said syndrome and complex.

DETAILED DESCRIPTION OF THE INVENTION AND OF THE PREFERRED EMBODIMENTS 
The compound found useful in the method of this invention is represented by 
formula I 
##STR5## 
including the pharmaceutically acceptable salts and esters thereof as well 
as the stereochemical isomers thereof. 
The compounds of formula I may form pharmaceutically acceptable acids such 
as hydrochloric, hydrobromic, methanesulfonic, toluenesulfonic, sulfuric 
or nitric or trihaloacetic acids, e.g. trichloroacetic and trifluoroacetic 
acids. The compounds of this invention of formula I also may form salts by 
reaction of the carboxylic acid novelty at C-5 with pharmaceutically 
acceptable bases such as the hydroxides of the alkali metal (especially 
Li, Na or K), the alkaline earth (esp Ca, Mg and Sr) or ammonium 
hydroxides. Pharmaceutically acceptable esters of compounds of formula I 
may be include C.sub.1 -C.sub.6 lower alkyl especially methyl and ethyl as 
well as p-nitrobenzyl, indanyl, phthalidyl, methoxymethyl, glycloxymethyl, 
phenylglycyloxymethyl, thienylglycyloxymethyl, acetoxymethyl and 
pivaloyloxymethyl. 
The compound represented by formula I includes stereo isomers due to the 
presence of two asymetric carbon atoms (C-2 and C-5) and geometric 
isomerism at (C.sub.9 and C.sub.10. All stereoisomers of formula I are 
contemplated as part of this invention. 
In a preferred aspect of the method of the present invention, it is 
preferred to use the stereoisomers of the compund of formula I having the 
formula II: 
##STR6## 
wherein the L indicates that L enantiomers of alanine and of 
2,3-diaminopropanoic acid and the hydrogens attached to C-9 and C-10 are 
trans relative to each other. 
The preferred stereoisomers of the compounds of formula I are represented 
by Formula II and are isolated from an antimicrobial (anti viral) complex 
produced by cultivating a strain of Micromonospora sp SCC 2217 having the 
identifying characteristics of ATCC 55064 in a pH and temperature 
controlled aqueous nutrient medium containing assimilable sources of 
nitrogen and carbon, under controlled submerged aerobic conditions, until 
a composition of matter having substantial antimicrobial activity is 
produced. 
A variable culture of the Micromonospora sp SCC 2217 ATCC 55064 was 
deposited on Jun. 12, 1990 with The American Type Culture Collection, 
12301 Parklawn Drive, Rockville Md. 20852 U.S.A. 
The stereoisomers represented by formula II may also be prepared by 
epoxidation of N.sup.2 -L-alanyl-N.sup.3 
-fumaramoyl-L-2,3-diaminopropionic acid which has been prepared by R. 
Andruszkiewicz et al [The Journal of Antibiotics (1984). Vol 37 (No. 11) 
pp 1479-1482]. Suitable epoxidation reagents include peroxy acids such as 
peracetic and pertrichloroacetic acids, metachloroperoxybenzoic acid as 
well as those disclosed by J. March in "Advanced Organic Chemistry", 
McGraw-Hill, 1968, N.Y., at pp 578-579 and 618-621. 
The racemic mixture or D-enantiomer of compounds represented by formula I 
may be prepared by substitution in the procedure of R. Andruszkiewic supra 
of racemic or D-alanine for L-alanine, respectively, and of racemic or 
D-2,3-diaminopropionic acid for the L-enantiomer thereof and of maleamic 
acid, respectively, for the fumaramic acid. 
Antiviral Activity 
A sample of the compound represented by Formula II was tested in vitro for 
activity against the HIV-IIIb strain of HIV-1 growing in MT-2 cells, a 
human lymphocyte derived cell line in accordance with the procedure 
disclosed by P. Daniels et al. in Antiviral Research SI (1990), 1-130 
(Elsevier, Science Publishers B.V.) (0166-3542/90). 
The sample was tested at a single concentration (1:20 dilution of the 
submitted broth), in duplicate on HIV-1 infected MT-2 cells to measure 
antiviral efficacy and at the same concentration on uninfected MT-2 cells 
to measure the cytotoxic effect of the product. Assays were set up in 
96-well plates, the sample was tested and three replicates of virus 
control cultures (untreated, infected cells) and three replicates of cell 
control cultures (untreated, uninfected cells) were used. A panel of 
control cultures (AZT and DDC at various concentrations; alone and in 
combination with the sample) were set up with each test. Assay end-points 
were read at Day 7 post-infection by MTT, a formazan-activating 
tetrazolium salt used to measure cell viabilities (Tata, et al, J. Immunol 
Methods, 93:157-165). Criteria for selection of active compounds included 
arbitrary limits of 49% or less viability of test sample=inactive; 50-74% 
viability=marginally active; and 75-100%=active. Active and marginally 
active compounds were re-tested to confirm activity. Compounds meeting 
criteria of active are undergoing further in vitro activity and 
cytotoxicity testing. 
The compound represented by Formula II reduces the cytopathogy (CPE) caused 
by HIV-IIIb strain of HIV-1 by 50% at a concentration of 10 .mu.g/m L. The 
% CPE reduction for the HIV-infected MT-2 cells is defined by the 
relationship: 
##EQU1## 
FIG. 1 graphically illustrates the in-vitro activity of the compound 
represented by formula II. 
The sole FIGURE graphically displays the in vitro activity of the preferred 
compound represented by formula II against MT-2 cells infected with the 
HIV-IIIb strain of HIV as well as the against the uninfected MT-2 cells at 
the six concentration of such preferred compound of 0.32, 1,3.2, 10, 32 
and 100 .mu.g/mL (noted on the horizontal axis of FIG. 1 ). The reduction 
in the cytopathology (CPE) opf the HIV-IIIb infected MT-2 cells, i.e. the 
antiviral activity, of the stereoisomer of the compound represented by 
formula II at the six concentrations is plotted versus the right vertical 
axis to give the curve defined by the data points in the form of boxes. 
The viability of cells uninfected by the HIV-IIIb strain at the same six 
contrations is plotted versus the left vertical axis to give the upper 
curve defined by the data points in the form of X's. 
At concentrations of the compound represented by formula II as high as 100 
.mu.g/mL, the uninfected MT-2 tissue culture cell remained 100% viable. 
Thus, the selectively index for the compound represented by formula II is 
at least 10 or more, i.e., the compound represented by formula II inhibits 
HIV-1 replication at concentraiton at least 10 times below the levels at 
which the viability of uninfected human lymphocyte cells are impaired. 
This assay is predictive of clinical utility in mammals infected with 
AIDS. See R. Pauwels et al. Nature (1990), Vol. 343, p 470-474. 
The methods and pharmaceutical compositions of the present invention are 
useful for treating mammals infected with the human immunodeficiency virus 
Type 1. The progression of HIV-1 in mammals, especially male and female 
human beings has been separated into roughly three stages: (1) the early 
or acute phase, lasting weeks; (2) the middle or chronic phase, lasting 
and characterized by minimal but measurable pathologic changes; and the 
final or crisis phase generally referred to as the AIDS-related complex 
(ARC) or AIDS and lasting months to years depending on the effacy and 
availabilty of treatment. The mammals to be treated for the HIV-1 
infection or AIDS or ARC in accordance with the present invention complete 
standardized questionnaires about the signs and symptoms of HIV infection, 
and are given a complete physical examination and are classified according 
to their stage of disease caused by HIV-1 as defined by the Centers for 
Disease Control, MMWR (1986), Vol. 35, Suppl. 15, pp. 334-9 and MMWR 
(1987), Vol. 36, Suppl. 15, pp. 35-155. The progress of the HIV-1 
infection and the evaluation of responses to treatment in accordance with 
this invention may be determined by measurement of frequency of isolation 
of HIV-1 in peripheral-blood mononuclear cells (PBMC) and the frequency of 
isolation of HIV-1 from cell-free plasma (plasma viremia) (3) the presence 
and titer of p24 antigen in plasma and (4) the presence and filter of 
antibody to p24 antigen in accordance with the methods disclosed by R. W. 
Coombs et al., N. Engl. J Med. (1989), Vol. 321 (No. 24), pp 1626-1631 and 
Dr. D. Ho et al., ibid (1989), Vol. 321 (No. 24), pp 1621-1625. Ho et al. 
and Coombs et al. disclose that the increasing levels of HIV-1 in plasma 
viremia provides a good indication of the clinical status of a mammal 
infected with HIV-1 and provides support for early treatment of the 
infection. The prognostic value of three cellular (the number of CD4+T 
cells, the number of CD8+T cells and the ratio of CD4+T cells to CD8 T 
cells) and five serologic markers (serum levels of neopterin--a product of 
stimulated macrophages), beta.sub.2 -microglobulin, soluble interleukin-2 
receptors, IgA and HIV p24 antigen with HIV-1 as well as the methods of 
measuring all eight markers are disclosed by J. L. Fahey et al. in the N. 
Engl. J. Med. (1990), Vol. 322 (No. 3), p 166-17. J. L. Fahey et al. 
disclose that the progression to AIDS was predicted in mammals infected 
with HIV-1 most accurately by the level of CD4+T cells in combination with 
the serum level of teither neopterin or beta.sub.2 -microglobulin. At 
least one of the aforesaid serum markers, which reflect immune activation, 
should be used along with measurement of CD4+T cells in HIV-1 cause 
disease classification schemes and in the evaluation of the response to 
treatments in accordance with the present invention. 
The presence of HIV-1 in mammals to be treated in accordance with the 
present invention may also be determined in apparently healthy persons by 
use of test of blood thereof for HIV-1 antibody by two different enzyme 
immunoassays and a Western blot assay, as disclosed by J. B. Jackson et 
al. in the N. Engl. J. Med. (1990), Vol. 322 (No. 4), pp 217-222. 
The "therapeutically effective amount" of a pharmaceutical acceptable salt 
or ester or stereoisomer of a compound represented by formula I is in a 
dosage range of about 1 to about 100 mg/kg (body weight) per day with 1 to 
about 10 mg per day being preferred. 
The precise amount of a composition containing a compound represented by 
formula I or salt or ester or stereoisomer thereof would be determined by 
the attending clinicians taking into account the etiology and severity of 
the disease, the patients' condition, sex and age and other factors. 
The compounds represented by formula I or salts or esters or stereoisomers 
thereof and the pharmaceutical compositions of the present invention may 
be administered by parenteral, oral, systemic or topical means. 
The compound represented by formula I or a pharmaceutically acceptable salt 
or ester or stereoisomer thereof may be compounded into a dosage form 
suitable for oral or parenteral administration. A tablet or capsule or 
caplets are particularly convenient forms for oral administration. Such 
compositions useful in the present invention are typically formulated with 
conventional pharmaceutical excipients, e.g., spray dried lactose and 
magnesium stearate into tablets or capsules for oral administration. One 
or more of the active substances, with or without additional types of 
active agents, can be worked into tablets or dragee cores by being mixed 
with solid, pulverulent carrier substances, such as sodium citrate, 
calcium carbonate or dicalcium phosphate, and binders such as polyvinyl 
pyrrolidone, gelatin or cellulose derivatives, possibly by adding also 
lubricants such as a magensium stearate, sodium lauryl sulfate, "Carbowax" 
or polyethylene glycols. Of course, taste improving substances can be 
added in the case of oral administration forms. 
As further forms of administration, one can use plug capsules, e.g. hard 
gelatin, as well as closed soft gelatin capsules comprising a softner or 
plasticizer, e.g., glycedne. The plug capsules contain the active 
substance preferably in the form of a granulate, e.g., in mixtures with 
fillers, such as lactose, saccharose, mannitol, starches such as potato 
starch or amylopectin, cellulose derivatives or highly-dispersed silicic 
acids. In soft-gelatin capsules, the active substance is preferably 
dissolved or suspended in suitable liquids, such as vegetable oils or 
liquid polyethylene glycols. 
The compounds represented by formula I used in accordance with the present 
invention may also be formulated into once-a-day or even longer sustained 
release composition by conventional techniques well known in the art. 
In place of oral administration, the compounds represented by formula I may 
be administered parenterally. In such case, one can use a solutin of the 
active substance, e.g., in sesame oil or olive oil. 
EXAMPLE 1 
a) FERMENTATION 
The culture of Micromonospora sp SCC 2217, ATCC 55064 was stored as a stock 
suspension at -20.degree. C. in a growth medium containing 12% (w/v) 
sucrose solution. Three mL of a thawed suspension was used to inoculate 70 
mL of growth medium consisting of the following ingredients listed in 
concentrations expressed as percent (%) by weight per unit volume: beef 
extract 0.3% (w/v), tryptone 0.5%, yeast extract 0.5%, cerelose 0.1%, 
potato starch 2.4%, CaCO.sub.3 0.2% and Dow-Corning antifoam-B 0.1%, in a 
250-mL Erlenmeyer flask. After 48 hours incubation at 30.degree. C. on a 
rotary shaker operating at 300 rpm, 25 mL of the resulting cell suspension 
was used to inoculate 500 ml of the growth medium in a 2-liter Erlenmeyer 
flask. The culture was incubated as described above. After 48 hours, the 
entire contents of the flask were used to inoculate 10 liters of 
fermentation medium consisting of NZ-amine A 0.5%, yeast extract 0.5%, 
cerelose 1.0%, soluble starch 2.0%, CaCO.sub.3 0.4%, COCl.sub.2 0.0004% 
and Dow-Corning antifoam-B 0.1% in a 14-liter fermentor (New Brunswick 
Scientific, Edison, N.J.). The fermentation was carried out at 30.degree. 
C. with aeration of 3.5 liters/minute and agitation of 350 rpm. The pH and 
dissolved oxygen levels were continuously monitored without adjustment 
during the entire fermentation by means of probes submerged in the vessel. 
Microbial growth was determined by packed cell volume. The antibiotic 
production started 48 hours after inoculation, then gradually increased 
reaching a maximum at 96 hours. The amount of antibiotic produced was 
determined by a paper-disk agar diffusion method using Candida albicans 
strain 406 as the test organism. 
b) ISOLATION AND PURIFICATION OF THE COMPOUND REPRESENTED BY FORMULA II 
The compound represented by formula II was recovered from 110 liters (pH 7) 
of fermentation broth filtrate obtained in accordance with the procedure 
Example 1 (a) of by absorption thereof on the cation exchange BioRad AG 
50.times.8 (H.sup.+). Elution with 2 liters of 0.5N NH.sub.4 OH provided a 
filtrate which was concentrated to give 78.8 g of eluate. The concentrated 
eluant was absorbed onto BioRad AG 1.times.8 (HCO.sub.3.sup.-) and eluted 
with CO.sub.2 -saturated water. The eluant was lyophilized and the 
resultant solid (3.4 g) was subjected to chromatography on charcoal and 
elution with 1 liter gradient 0 to 20% aq. MeOH to provide 1.3 g of a 
white amorphous solid having the following physico-chemical properties: 
Physico-Chemical Properties of the Compound Represented by Formula II 
Amphoteric, water soluble compound, MP 198.degree. C. (dec) 
[.alpha.].sup.26.sub.D -30.6.degree. (c.0.5, H.sub.2 O) 
Stable at RT in the pH 2 to 9 range. 
FAB Mass Spectra Data: 
MW: 288 amu 
M+H: m/z 289 (found 289.1163, calc. 289.1148) corresponding to the 
molecular formula C.sub.10 H.sub.17 N.sub.4 O.sub.6. 
UV.lambda. .sub.(max) (H.sub.2 O): no absorbance maxima greater than 215 
nm. 
IR (KBr): 3300 cm.sup.-1 (br, NH, and OH) and 1650 cm.sup.-1 (amide). 
.sup.13 C NMR showed the presence of 10 carbon atoms including four 
carbonyls; the two carbon signals at 54.2 and 54.6 ppm were assigned to 
the epoxide carbons. i.e. C-.sub.9 and C-.sub.10 in formula II. 
.sup.1 H NMR spectrum in D.sub.2 O was also consistent with the presence of 
an epoxide and two epoxide protons having the trans configuration relative 
to each other on the basis of the small coupling constant. 
Acid hydrolysis of the white amorphous solid of Example 1b gave L-alanine 
and L-2,3-diaminopropanoic acid. The above physico-chemical data and the 
pH-dependent .sup.1 H NMR chemical shift study is consistent with the 
structure shown by formula II. [See R. Cooper et al., The Journal of 
Antibiotic Vol. 41 (No. 1), pp 13-19.]