Method of inhibiting virus

A method of inhibiting human immunodeficiency virus is disclosed which comprises administering to a patient infected with said virus a virally inhibitory effective amount of the N-butyl derivative of deoxynojirimycin or a pharmaceutically acceptable salt derivative thereof.

BACKGROUND OF THE INVENTION 
This invention relates to a method of inhibiting human immunodeficiency 
virus (HIV) and, more particularly, to the N-butyl derivative of 
1,5-dideoxy-1,5-imino-D-glucitol (deoxynojirimycin) having potential use 
for the treatment of acquired immune deficiency syndrome (AIDS). 
Acquired immune deficiency syndrome, which only a few years ago was a 
medical curiosity, is now a serious disease. As a consequence, a great 
effort is being made to develop drugs and vaccines to combat AIDS. The 
AIDS virus, first identified in 1983, has been described by several names. 
It is the third known T-lymphocyte virus (HTLV-III) and has the capacity 
to replicate within cells of the immune system and thereby lead to a 
profound destruction of T4.sup.+ T-cells (or CD4.sup.+ cells). See, e.g., 
Gallo et al., Science 224, 500-503 (1984), and Popovic et al., Ibid., 
497-500 (1984). This retrovirus had been known as 
lymphadenopathy-associated virus (LAV) or AIDS-related virus (ARV) and, 
most recently, as human immunodeficiency virus (HIV). Two distinct AIDS 
viruses, HIV-1 and HIV-2, have been described. HIV-1 is the virus 
originally identified in 1983 by Montagnier and co-workers at the Pasteur 
Institute in Paris [Ann. Virol. Inst. Pasteur 135 E, 119-134 (1984)], 
while HIV-2 was more recently isolated by Montagnier and his coworkers in 
1986 [Nature 326, 662 (1987)]. As used herein, HIV is meant to refer to 
these viruses in a generic sense. 
Although the molecular biology of AIDS is beginning to be unraveled and 
defined, much more needs to be learned and understood about this disease. 
In the meantime, numerous approaches are being investigated in the search 
for potential anti-AIDS drugs and vaccines. Development of an AIDS vaccine 
is hampered by lack of understanding of mechanisms of protective immunity 
against HIV, the magnitude of genetic variation of the virus, and the lack 
of effective animal models for HIV infection. See, for example, Koff and 
Hoth, Science 241, 426-432 (1988). 
The first drug to be approved by the U.S. Food and Drug Administration 
(FDA) for treatment of AIDS was zidovudine, better known under its former 
name azidothymidine (AZT). Chemically, this drug is 
3'-azido-3'-deoxythymidine. This drug was originally selected as a 
potential weapon against AIDS because it was shown to inhibit replication 
of the virus in vitro. Such in vitro tests are useful and virtually the 
only practical method of initially screening and testing potential 
anti-AIDS drugs. A serious drawback of AZT, however, is its toxic 
side-effects. Thus, the search for better anti-AIDS drugs continues. 
More recently, certain glycosidase inhibitors have been tested for activity 
against the AIDS virus. Three such compounds suggested as potential 
anti-AIDS drugs are castanospermine, 1-deoxynojirimycin (DNJ) and 
2,5-dihydroxymethyl-3,4-dihydroxy-pyrrolidine (DMDP). See, e.g., Sunkara 
et al., Biochem. Biophys. Res. Commun. 148(1), 206-210 (1987); Tyms et 
al., Lancet, Oct. 31, 1987, pp. 1025-1026; Walker et al., Proc. Natl. 
Acad. Sci. USA 84, 8120-8124(1987); and Gruters et al., Nature 330, 74-77 
(1987). 
##STR1## 
Thus, castanospermine, which is an alkaloid isolated from the seeds of 
Australian chestnut tree, has been found to interfere with normal 
glycosylation of HIV virions, thereby altering the envelope glycoprotein 
and preventing entry of HIV into target cells. However, only a modest 
reduction in virion infectivity was found. 
In PCT Inter. Appln. WO 87/03903, published July 2, 1987, the N-methyl 
derivative of deoxynojirimycin (DNJ) also was dictated as having activity 
against HIV ostensibly based on its glucosidase I inhibitory activity. 
However, it was subsequently shown by Fleet et al., FEBS Lett, In Press, 
1988, that not all glucosidase I inhibitors are effective inhibitors of 
HIV. Therefore, some other mechanism may be responsible for HIV inhibitory 
activity. 
BRIEF DESCRIPTION OF THE INVENTION 
In accordance with the present invention the N-butyl derivative of 
deoxynojirimycin has been found to have substantially enhanced inhibitory 
activity against the human immunodeficiency virus (HIV) at non-toxic 
concentrations compared to that exhibited by the corresponding N-methyl 
and N-ethyl derivatives. The N-butyl-deoxynojirimycin uniquely reduces the 
virus titer by over five logs at non-cytotoxic concentrations whereas the 
N-methyl- and N-ethyldeoxynojirimycin derivatives cause only a two to four 
log-order of reduction in the yield of infectious HIV. As such, the 
N-butyl derivative has exceptional and significant potential use for the 
treatment of acquired immune deficiency syndrome (AIDS). This outstanding 
HIV inhibitory activity was surprising and unexpected, if and only if 
glucosidase I inhibitory activity is the operative mechanism, since the 
glucosidase I inhibitory activity of the N-methyl and N-butyl derivatives 
of deoxynojirimycin was reported to be virtually identical by Schweden et 
al., Arch. Biochem. Biophys. 248(1), 335-340 (1986). 
The N-butyl derivative of deoxynojirimycin has the following chemical 
structure: 
##STR2## 
In order to indicate stereoisomerism, solid and dotted lines show bonds 
directed above or below, respectively, the plane of the paper.

The N-butyl derivative of deoxynojirimycin is a known compound. It can be 
prepared by the N-butylation of 1,5-dideoxy-1,5-imino-D-glucitol 
(deoxynojirimycin). Methods of preparation are described, for example, in 
U.S. Pat. Nos. 4,182,767 and 4,639,436. 
The effectiveness of the active N-butyldeoxynojirimycin in the method of 
the invention has been demonstrated by positive inhibitory activity toward 
replication of the HIV in vitro. In accordance with this assay system, 
human T cells which are susceptible to HIV infection were used to visually 
determine the relative activity of several test compounds to inhibit 
replication of the HIV infected cells. Since various analogous compounds 
had substantially divergent results as described hereinafter, it is 
apparent that the effectiveness of any given compound as an inhibitor of 
HIV is unpredictable. Various theories have been proposed heretofore with 
respect to the effect of prior art HIV inhibitors. Research at several 
laboratories has established that interaction between the envelope 
glycoprotein, gp120, and some part of the CD4 antigen is involved in the 
recognition of HIV and most of the cells it infects and the binding of HIV 
to those cells. Thus, in one report which compared the positive effect of 
the glycosidase inhibitor deoxynojirimycin (DNJ) upon HIV infectivity 
against the absence of effect of the mannosidase I inhibitor 
deoxymannojirimycin (DMJ), which is the 2-epimer of DNJ, it was suggested 
that the perturbed carbohydrate structure of the gp120 or its precursor, 
imposed by the blocking of the N-linked oligosaccharide trimming pathway, 
is responsible for the effect. Gruters et al., Nature 330, 74-77 (1987). 
The unpredictable effect of a test compound against HIV is demonstrated by 
several comparative studies of structurally analogous sugar derivatives. 
For example, while the known inhibition of the cytopathic effect (CPE) by 
the .alpha.-glucosidase I inhibitor castanospermine is confirmed, neither 
the epimer L-1, 6-diepicastanospermine nor the stereoisomer of 
castanospermine, L-6-epicastanospermine, were found to be inhibitory. See 
Fleet et al., FEBS Lett., In Press, 1988. 
So also, although both enantiomers of 1,4-dideoxy-1,4-imino-arabinitol are 
known glucosidase inhibitors [Fleet et al., Tetrahedron Lett. 26, 
3127-3130(1985); Fleet et al., Chemistry Lett. 1051-1054(1986)]; the 
L-enantiomer has strong HIV inhibitory activity [see also copending 
application Ser. No. 136,219, filed Dec. 21, 1987] whereas the 
D-enantiomer has very little effect on HIV replication. For both 
enantiomers, N-methylation reduced rather than increased anti-HIV 
activity. Neither the azofuranose analog of glucose nor the N-benzyl 
derivative were found to have an effect on CPE. Similarly, no HIV 
inhibition was observed for fagomine, the 2-deoxyglucose analog, although 
it too is known to have .alpha.-glycosidase inhibitory activity. See Fleet 
et al., FEBS Lett., In Press, 1988. 
The inhibitory activity of the N-butyldeoxynojirimycin of this invention 
toward HIV relative to the inhibitory activity of analogous test compounds 
is specifically demonstrated herein by an in vitro assay system in which 
T-cells are grown in suitable nutrient culture medium and exposed to HIV 
inoculum in the presence or absence of test compound and compared with 
control cells which are grown in culture medium alone. After a suitable 
period of incubation, the cultures are scored for the presence of 
so-called syncytial cells (giant cells). Typical examples of such a test 
for the evaluation of inhibitors of HIV have been disclosed by Fung et 
al., Bio/Technology 5, 940-946 (1987); Tyms et al., Lancet, Oct. 31, 1987, 
pp. 1025-1026; Gruters et al., Nature 330, 74-77 (1987); and Walker et 
al., Proc. Natl. Acad. Sci. USA 84, 8120-8124 (1987). 
In the present case, a human leukemic T-cell line was used which is 
described by Karpas, Leuk. Res. 1, 35-49 (1977). This cell line (T-45) was 
established from a child with acute lymphoblastic leukemia. Another T-cell 
line used to demonstrate the inhibitory activity of the 
N-butyl-deoxynojirimycin is the MOLT-4 cell line. This cell line was 
originally derived from the peripheral blood of a patient with acute 
lymphoblastic leukemia. Further information on the origin and 
characteristics of this cell line can be had by reference to Minowada, J. 
Natl. Cancer Inst. 49, 891-895 (1972). The MOLT-4 cell line is on deposit 
without restriction in the permanent collection of the American Type 
Culture Collection, Rockville, Maryland, under accession number ATCC CRL 
1582. Samples of the cell line can be obtained by the public upon request 
to that depository. 
Cell free suspensions of the HIV were prepared from infected cultures. The 
concentration of infectious particles was estimated on end-point titration 
assay using serial ten-fold dilutions. The approximate number of 
infectious particles in each preparation was determined by the highest 
dilution which contains infectious HIV as determined by synctial 
formation, cytophaticity [Leonard et al., Proc. Natl. Acad. Sci. USA 85, 
3570-3574 (1988); Barre-Sinoussi et al., Science 220, 868-870 (1983)] and 
HIV antigen [Karpas et al., Lancet 1, 695-697 (1985)] synthesis after 10 
days of culture with 10.sup.4 T-cells. This represents the concentration 
of infectious particles, herein defined as the tissue culture infectious 
dose (TCID). 
The assay was run in 96 well plastic microtiter plates in which 0.2 ml of 
culture medium containing 10.sup.4 T-cells are seeded into each well. 
RPMI-1640 supplemented with 10% fetal calf serum was used as the culture 
medium. In each assay, 6 wells are used. Three (1-3) are infected with 
10.sup.4 TCID/well of HIV-1 or HIV-2 and to the other three (4-6) culture 
medium alone is added. Medium is changed at the same intervals for each 
test compound. The detailed assay procedure is illustrated in FIGS. 1 and 
2. 
The following examples will illustrate the invention in greater detail 
although it will be understood that the invention is not limited to these 
specific examples or the details therein. 
EXAMPLE 1 
The HIV inhibitory activity of N-n-butyldeoxynojirimycin (BuDNJ) was 
assessed in various comparisons with the N-methyl (MeDNJ) and N-ethyl 
(EtDNJ) analogs and with castanospermine (Cast), 
1,4-dideoxy-1,4-imino-L-fucitol (LAB) and 
N-(5-carboxymethyl-1-pentyl)-1,5-imino-L-fucitol (LFT), and other 
aminosugar derivatives mentioned hereinbefore. 
The efficacy of the test compounds used in this example was assessed as 
follows: 0.2 ml of culture medium containing 10.sup.4 T-45 cells were 
transferred into each well of a 96-well flat-bottomed tissue-culture 
plastic plate and the cells were allowed to settle at 37.degree. C. (see 
FIG. 1). After 4 hours the media were aspirated and replaced with media 
which contained the test compound. Following overnight incubation the 
media was aspirated and 10.sup.4 TCID of virus (HIV-1 or HIV-2) was added 
to each well. Incubation was continued at 37.degree. C. in 5% CO.sub.2 for 
1 hour. Thereafter, growth media containing the various compounds were 
added and incubation at 37.degree. C. was continued. Control cultures were 
maintained as indicated in FIG. 2. On the fourth day the cell suspensions 
from each well were split and seeded into two new wells and 0.2 ml of 
fresh media with compounds was added. This was repeated on the seventh 
day. Cells were examined by microscopy during days 1 to 10 for the 
presence of syncytia, growth rate and the appearance of the ctyopathic 
effect (CPE) (giant cells, pycnotic nuclei, loss of refractility). CPE 
(100%) was defined as no round, refractile, uniform cells remaining; only 
giant, ballooned pyknotic cells were present. The viability of HIV 
infected cultures was determined by a rough count/estimate of cells which 
underwent CPE in relation to round refractile uniform (normal) cells in a 
given culture. Cells in wells which were incubated with compounds which 
appeared to inhibit or reduce HIV replication and in which there was 
marked cell proliferation were either transferred to larger wells (of a 
24-well plate) or divided in order to maintain an approximate constant 
cell density to promote continuous cell division. 
To determine if there was a gradual reduction of HIV-infected cells in 
cultures maintained with BuDNJ, aliquots of cells infected with 10.sup.4 
TCID of HIV-1 and grown in the presence of 0.1 mg/ml BuDNJ for varying 
lengths of time were transferred in separate wells and grown in drug-free 
medium as indicated in FIG. 3. The cultures were then monitored for 
development of CPE, indicative of active HIV replication. The times for 
the appearance of an advanced CPE were recorded. To confirm that HIV-1 or 
HIV-2 was the cause of the CPE, cells were fixed on glass slides to verify 
the expression of the corresponding viral antigen using the method 
described by Karpas et al., Lancet 1, 695-697 (1985). Compounds which were 
found to inhibit HIV-1 and HIV-2 replication in T-45 cells were 
subsequently used with the MOLT-4 human leukaemic T-cell line. 
The results of the foregoing tests were as follows: 
The effect of various concentrations of MeDNJ, EtDNJ, Cast, BuDNJ, LFT and 
LAB, respectively, on CPE formation as shown in FIG. 3. These data show a 
bell shaped dose-dependence for MeDNJ, EtDNJ and Cast. The cell viability 
data shown in FIG. 3 and growth rates in Table 1 for non-infected cells 
grown in the presence of MeDNJ, EtDNJ and Cast suggest that these 
compounds lack selective anti-viral activity, and are cytotoxic, as would 
be expected for inhibitors of oligosaccharide biosynthesis. Therefore, the 
apparent loss of anti-viral activity (bell shaped dose-dependence) of 
these drugs at high concentrations arises from the cytotoxic effect of the 
drugs mimicking and being scored as an HIV induced cytopathic effect. In 
contrast, the compounds BuDNJ, LFT and LAB showed no cytotoxicity over the 
concentration range used. BuDNJ completely prevented CPE in the HIV 
infected cells at all concentrations tested. The difference in behavior 
between BuDNJ and the other alkyl analogues of DNJ, (MeDNJ and EtDNJ), 
suggests that its mechanism of action may be different. Similarly, the 
.alpha.-fucosidase inhibitor LFT which doesn't disrupt oligosaccharide 
biosynthesis was not found to be cytotoxic. The properties of LAB were 
similar to LFT. The activity of LAB as an .alpha.-glucosidase inhibitor is 
reported by Fleet et al., FEBS Lett., In Press, 1988, but has yet to be 
tested for activity against the processing .alpha.-glucosidases (i.e. I 
and II). The HIV inhibitory activity of LFT and LAB is disclosed in 
corresponding application Ser. No. 136,219, filed Dec. 21, 1987. 
Quantitative data on the effect of the various compounds on the 
non-infected cells (cytotoxicity) was obtained by comparison of their 
growth rate to control cells not exposed to drugs (Table 1). FIG. 4 and 
Table 2 show the relation between the TCID titre of culture supernatants 
of T-45 cells infected with HIV after 10 days in culture and the 
concentration of drug. These data show that LAB and LFT were only able to 
reduce the TCID partially, even at very high concentration of the drug. 
Similarly, DNJ, MeDNJ and EtDNJ reduced the TCID only partially, although 
to a greater extent than LAB and LFT. The inability of any of these drugs 
alone to totally reduce the TCID may be due to heterogeneity of virus 
production or spread. FIG. 4 and Table 2 shows that only BuDNJ was able to 
achieve negligible virus TCID titres at concentrations which were 
non-cytotoxic. These data together with the data presented in FIG. 3 
suggest a viral-specific activity for this compound. Table 2 shows the 
results of a direct side by side comparison of the anti-viral activity 
(reduction in TCID) of DNJ and its derivatives against HIV-1 and HIV-2. In 
addition, the MOLT-4 cell line was also used. These data show that similar 
anti-viral activity is found against HIV-1 and HIV-2 and that this 
activity is not restricted to virus grown in the T-45 cell line. 
In order to determine whether or not there was an actual reduction in 
HIV-infected cells in the presence of BuDNJ, aliquots of the infected cell 
suspension, grown for varying times in the presence or drug, where 
transferred to drug-free medium as outlined in FIG. 2. The cultures were 
then monitored for the development of CPE and giant cell formation 
(indicative of active HIV replication). The increase in the time taken for 
the cells to develop CPE once drug-free medium was used (FIG. 5) suggests 
that prolonged exposure to BuDNJ reduced the proportion of infected cells 
in the cultures. Whether this is viewed as either a reduction in the 
doubling time of the infected cells as compared to uninfected T-45 cells 
or as viral replication with cytolysis does not alter the conclusion that 
natural turnover of the cell population in vivo would eventually reduce 
the number of infected cells dramatically and possibly break the cycle of 
re-infection. 
TABLE 1 
______________________________________ 
CYTOTOXICITY AND T-CELL GROWTH 
Estimated Cell Growth 
Dosage Virus-uninfected 
Compound (mg/ml) Day 7 
______________________________________ 
No drug -- 1.2 .times. 10.sup.6 
DNJ 0.50 1.4 .times. 10.sup.6 
0.25 1.4 .times. 10.sup.6 
MeDNJ 0.50 5.0 .times. 10.sup.6 
0.25 1.0 .times. 10.sup.6 
0.10 1.0 .times. 10.sup.6 
0.05 1.2 .times. 10.sup.6 
0.01 1.2 .times. 10.sup.6 
EtDNJ 0.10 1.3 .times. 10.sup.6 
0.05 1.2 .times. 10.sup.6 
0.01 1.2 .times. 10.sup.6 
BuDNJ 0.10 1.4 .times. 10.sup.6 
0.05 1.2 .times. 10.sup.6 
0.01 1.2 .times. 10.sup.6 
LAB 0.50 1.0 .times. 10.sup.6 
0.25 1.5 .times. 10.sup.6 
0.10 1.5 .times. 10.sup.6 
Cast 0.70 Toxic 
0.35 8.0 .times. 10.sup.5 
0.18 8.0 .times. 10.sup.5 
0.09 1.0 .times. 10.sup.6 
0.02 1.2 .times. 10.sup.6 
______________________________________ 
TABLE 2 
______________________________________ 
COMISION OF DNJ DERIVATIVES 
Dosage HIV-1 HIV-2 
Virus (mg/ml) (TCID) (TCID) 
______________________________________ 
Control -- 10.sup.6 10.sup.6 
DNJ 0.10 10.sup.5 10.sup.5 
MeDNJ 0.10 10.sup.2 10.sup.2 
EtDNJ 0.10 10.sup.3 10.sup.3 
BuDNJ 0.10 &lt;10 .sup. 
&lt;10 .sup. 
______________________________________ 
Identical results were found when either 10.sup.4 T45 cells or MOLT4 
cells were treated with DNJ and its three derivatives before infecting th 
cells with HIV1 and separately with HIV2 (10.sup.4 TCID). The HIV1, which 
was used to infect the T45 cell line was first passaged in T45 cells. 
Likewise, HIV1 which was used to infect MOLT4 was first passaged in MOLT4 
cells. The same was done with HIV2. TCID's were measured after day 10. 
EXAMPLE 2 
In order to particularly illustrate the HIV inhibitory activity of 
N-butyl-deoxynojirimycin of the invention compared to the activity of the 
N-methyl and N-ethyl analogs as well as the non-alkylated deoxynojirimycin 
and untreated controls, the following tabulation shows the results of 
tests carried out according to the procedure of Example 1 on both T-45 
cells and MOLT-4 cells with HIV-1 and, separately, with HIV-2. That is, 
HIV-1 was first passaged in T-45 cells; likewise, HIV-1 was first passaged 
in MOLT-4 cells. The same passaging was done with HIV-2. The test 
compounds were used at a concentration of 0.1 mg/ml. 10.sup.4 TCID of 
HIV-1 and separately of HIV-2 were used to infect 10.sup.4 cells. Upon 
completion of the test, the virus titers were as follows: 
TABLE 3 
______________________________________ 
VIRUS TITER 
Control DNJ MeDNJ EtDNJ BuDNJ 
______________________________________ 
HIV-1 10.sup.6 10.sup.5 
10.sup.2 
10.sup.3 
10 
HIV-2 10.sup.6 10.sup.5 
10.sup.2 
10.sup.3 
10 
______________________________________ 
These results thus show that the N-butyl-deoxynojirimycin surprisingly is 
not only two log orders more effective as an inhibitor of HIV than the 
N-methyl-deoxynojirimycin but also three log orders more effective than 
the N-ethyl deoxynojirimycin in replicate tests (Tables 2 and 3) each with 
two virus strains in two different cell lines. 
EXAMPLE 3 
The N-butyl-DNJ inhibitor of the invention was further differentiated from 
DNJ and N-methyl-DNJ by comparing the incorporation of radiolabeled 
galactose, mannose, glucose and thymidine into B16-F10 murine melanoma 
cells following incubation of the cells with the test compound. The 
following results were obtained: 
1. DNJ and N-butyl-DNJ each inhibited mannose utilization whereas 
N-methyl-DNJ either stimulated incorporation or, conservatively, had no 
effect. 
2. DNJ and N-butyl-DNJ each substantially inhibited galactose incorporation 
into the cells at 1.0 mM or less whereas N-methyl-DNJ was only slightly 
inhibitory. 
3. In glucose utilization/inhibition testing the N-methyl-DNJ had an 
inverted dose-response curve whereas N-butyl-DNJ had little or no effect 
and DNJ inhibited in a normal dose-response manner. 
4. .sup.3 H-Thymidine uptake into the cells was not affected by N-butyl-DNJ 
up to 2.0 mM whereas N-methyl-DNJ inhibited incorporation at 1.0 mM and 
DNJ inhibited incorporation at 2.0 mM. 
The antiviral agent described herein can be used for administration to 
patients infected with the human immunodeficiency virus by conventional 
means, preferably in formulations with pharmaceutically acceptable 
diluents and carriers. This agent can be used in the free amine form or in 
its salt form. Pharmaceutically acceptable salt derivatives are 
illustrated, for example, by the HCl salt. The amount of the active agent 
to be administered must be an effective amount, that is, an amount which 
is medically beneficial but does not present toxic effects which overweigh 
the advantages which accompany its use. It would be expected that the 
adult human dosage would normally range upward from about one milligram of 
the active compound. The preferable route of administration is orally in 
the form of capsules, tablets, syrups, elixirs and the like, although 
parenteral administration also can be used. Suitable formulations of the 
active compound in pharmaceutically acceptable diluents and carriers in 
therapeutic dosage form can be prepared by reference to general texts in 
the field such as, for example, Remington's Pharmaceutical Sciences, Ed. 
Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, PA. 
Various other examples will be apparent to the person skilled in the art 
after reading the present disclosure without departing from the spirit and 
scope of the invention. It is intended that all such further examples be 
included within the scope of the appended claims.