Method for treating retroviral infections with aryl-(2-pyrryl) ketone compound

A method for treating viral infections in a patient with aryl-(2-pyrryl) ketone compound and compositions.

TECHNICAL FIELD 
The instant invention comprises treating a patient infected with a 
retrovirus comprising administering to the patient a aryl-(2-pyrryl) 
ketone compound and compositions 
BACKGROUND OF THE INVENTION 
According to the World Health Organization there are currently 96,000 cases 
of AIDS reported worldwide. It is estimated that by 1992 the number of 
cases will have increased to 1.2 million. AIDS and AIDS Related Complex 
(ARC) are caused by infection with retrovirus designated HIV which 
includes the subtypes HIV-1 and HIV-2. HIV exerts a profound cytopathic 
effect on the CD4+ helper/inducer T-cells, devastating the function of the 
immune system. HIV infection also results in neurological deterioration 
and ultimately death of the infected individual. The field of viral 
chemotherapeutics has developed in response to the need for agents 
effective against retroviruses, particularly HIV. There are many ways in 
which an agent can exhibit anti-retroviral activity, one of which is 
through inhibition of viral replication. For example the HIV virus 
requires at least four viral proteins for replication: Reverse 
Transcriptase (RT), protease, the transactivator protein TAT, and the REV 
protein. Anti-retroviral agents such as AZT or ddC are known to be RT 
inhibitors. Anti-viral agents such as TAT inhibitors would act at a 
different stage of the viral life cycle. HIV replication in latently 
infected CD4+lymphocYtes is induced when the cells are stimulated to 
proliferate by cytokines or mitogens. The viral switch from latency to 
active replication requires the regulatory gene products TAT and REV. The 
TAT protein transactivates the HIV-LTR promoter and amplifies viral 
replication many thousand fold. The TAT responsive sequence is mapped 
within the LTR sequence. Compounds which have anti-TAT activity will thus 
arrest HIV at the latent stage of viral infection by preventing 
replication of the provirus that is integrated into the host cell 
chromosome. Anti-TAT agents are thus useful for therapeutically treating 
patients infected with HIV including AIDS and ARC patients or asymptomatic 
carriers. 
SUMMARY OF THE INVENTION 
The instant invention is directed to a method for treating a patient 
infected with a retrovirus, particularly HIV, comprising administering to 
the patient an anti-virally effective amount of a compound or a 
composition containing a compound of the formula: 
##STR1## 
The instant invention is also directed to a method for alleviating the 
cytopathic destructive effects of retroviral disease in a patient infected 
with a retrovirus comprising administering to said patient an anti-virally 
effective amount of Compound II or compositions containing such Compound 
II. 
The instant invention also includes anti-viral compositions comprising a 
therapeutically effective amount of Compound II in a pharmaceutically 
acceptable carrier.

DETAILED DESCRIPTION 
The compound which is used in the method of the invention (Compound II) is 
2-glycinamido-5-chlorophenyl (2-pyrryl) ketone. This Compound and its 
synthesis is disclosed in U.S. Pat. No's. 3,405,122; 3,398,159; 3,407,211; 
and 3,400,128 all of which are hereby incorporated by reference. Compound 
II exhibit anti-retroviral, particularly anti-HIV activity. 
Compound II is an anti-viral agent which exhibits anti-TAT activity as set 
forth in Example 1 and FIGS. I-II. This compound also exhibits anti-HIV 
activity as demonstrated by its inhibition of HIV-cytopathic effect and 
viral antigen production as set forth in Example 2. 
The instant invention is directed to treating patients infected with a 
retrovirus, comprising administering to the patient an anti-virally 
effective amount of Compound II or a composition containing Compound II. 
Patient in the context of the invention means human being and retroviral 
infection particularly includes HIV infection, including AIDS or ARC 
patients. An anti-virally effective amount of Compound II for treating a 
retroviral infection is in the range of 0.1 to 10 mg/kg body weight per 
day. This dosage may be administered in one or more doses at various 
intervals such as 2, 4, 6, 8, 12 or 24 hours. The suitable dosage is one 
that achieves a therapeutic blood level of 0.05-10 .mu.M, 0.1 to 5 .mu.M 
is preferred. This blood level may be best achieved by administering 
approximatelY 1-3 mg/kg body weight once or twice per day. 
The compounds may be administered in various dosage forms as set forth 
herein. Either the compounds, compositions, or their pharmaceutically 
acceptable salts are suitable. Pharmaceutically acceptable salts may be 
salts of organic acids such as lactic, acidic, malic, or p-toluenesulfonic 
acid and the like as well as salts of pharmaceutically acceptable mineral 
acids such as hydrochloric and sulfuric acids and the like. 
The compounds are administered in the dosages as set forth herein until 
alleviation of the retroviral infection. The compounds may also be 
administered with other anti-retroviral agents and particularly with known 
reverse transcriptase (RT) inhibitors such as ddC, AZT, ddI, ddA, or other 
inhibitors which act against other HIV proteins. Treatment with both 
anti-TAT agents such as Compounds I and II, and anti-RT agents should 
inhibit most or all replicative stages of the viral life cycle. The 
dosages of ddC and AZT used in AIDS or ARC patients have has been 
published. A virustatic range of ddC is generally between 0.05 .mu.M to 
1.0 .mu.M. A range of about 0.005-0.25 mg/kg body weight is virustatic in 
most patients. The preliminary dose ranges for oral administration are 
somewhat broader, for example 0.001 to 0.25 mg/kg given in one or more 
doses at intervals of 2, 4, 6, 8, 12, etc. hours. Currently 0.01 mg/kg 
body weight ddC given every 8 hours is preferred. When given in combined 
therapy the anti-RT compound may be given at the same time as Compound II 
or the dosing may be staggered as desired. The two drugs may also be 
combined in a composition. Doses of each may be less when used in 
combination then when they are used as a single agent. 
The instant invention is also directed to compositions containing a 
therapeutically effective amount of Compound II in a pharmaceutically 
acceptable carrier. It is possible for the Compounds of the invention to 
be administered alone in solution. However, it is preferred that the 
active ingredients be administered in a pharmaceutical formulation. In the 
context of the instant invention formulation means composition. These 
formulations comprise at least one active ingredient together with one or 
more pharmaceutically acceptable carriers and/or other therapeutic agents, 
for example an RT inhibitor. As included within the scope of this 
invention, "acceptable" is defined as being compatible with other 
ingredients of the formulation and not injurious to the patient or host 
cell. These carriers include those well known to practitioners in the art 
as suitable for oral, rectal, nasal, topical, buccal, sublingual, vaginal, 
or parenteral (including subcutaneous, intramuscular, intravenous, and 
intradermal) administration. Compound II may be used in the manufacture of 
pharmaceuticals for the treatment or prophylaxis of viral infections. The 
compositions may be conveniently presented in unit dosage form and 
prepared by methods known in the pharmaceutical art. Such methods include 
the preparation of the active ingredient in a carrier which may contain 
additional medicinally active ingredients, for example, Compound II in 
conjunction with a known RT inhibitor such as ddC or AZT. The compositions 
of the invention suitable for oral administration may consist of liquid 
solutions such as an effective amount of the compound dissolved in 
diluents such as water, saline, or orange juice. Capsules, sachets or 
tablets, each containing a pre-determined amount of the active ingredient, 
as a solid or granules; as a solution or suspension in an aqueous liquid; 
in an oil-in-water emulsion or a water-in-oil liquid emulsion, for 
example, soft gelatin capsules Tablet forms may include one or more of 
lactose, microcrystalline cellulose, colloidal silicon dioxide, 
croscarmellose sodium, magnesium stearate, stearic acid and other 
excipients, colorants, and pharmacologically compatible carriers. 
Formulations suitable for topical administration include lozenges 
comprising the active ingredient in a flavor, usually sucrose and acacia 
or tragacanth; pastilles comprising the active ingredient in an inert 
basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes 
comprising the active ingredient in a suitable liquid carrier. 
Formulations for rectal administration may be presented as a suppository 
with a suitable base comprising, for example, cocoa butter or a 
salicylate. 
Formulations suitable for vaginal administration may be presented as 
pessaries, tampons, creams, gells, pastes, foams, or spray formulas 
containing in addition to the active ingredient such carriers as are known 
in the art to be appropriate. 
Formulations suitable for parenteral administration include aqueous and 
non-aqueous, isotonic sterile injection solutions which may contain 
anti-oxidants, buffers, bacteriostats and solutes which render the 
formulation isotonic with the blood of the intended recipient; and aqueous 
and non-aqueous sterile suspensions which may include suspending agents 
and thickening agents. The formulations may be presented in unit-dose or 
multi-dose sealed containers, for example, ampules and vials, and may be 
stored in a freeze-dried (lyophilized) condition requiring only the 
addition of the sterile liquid carrier, for example, water for injections, 
immediately prior to use. Extemporaneous injection solutions and 
suspensions may be prepared from sterile powders, granules and tablets of 
the kind previously described. In the context of the invention formulation 
means composition. 
The instant invention is also directed to a method for alleviating the 
cytopathic destructive effects in a patient infected with a retrovirus 
comprising administering an anti-virally effective amount of the compounds 
or compositions of the invention. Since Compound II is known to inhibit 
TAT it can inhibit viral replication at the latent stage. The dosages 
mentioned previously would be suitable for this purpose. Preferably 1-3 
mg/kg body weight given once or twice a day provides the virustatic range 
of 0.01-5.0 .mu.M. The compounds can be administered to AIDS patients, ARC 
patients or asymptomatic HIV-infected patients. 
EXAMPLE 1 
Compound II was tested for anti-TAT activity in an anti-TAT assay described 
in copending patent application Case Docket No. 8320 filed on the same day 
as the instant application and hereby incorporated by reference. At 24 
hours post transfection 1, 10, and 100 .mu.M of test compound was added to 
the culture media of COS cells transfected with two plasmids, one 
containing the reporter gene which codes for Secreted Alkaline Phosphatase 
(SeAP) under control of HIV-LTR, and other containing the TAT gene also 
under control of HIV-LTR. The alkaline phosphatase activity of the media 
was assayed 48 hours after addition of test compound. The results are set 
forth in FIG. 1 which represents three independent assays of the test 
compound. The anti-TAT activity is measured by the percent inhibition of 
SeAP gene expression under the control of HIV-LTR. The cytotoxicity of the 
compound was tested in a parallel assay in which SeAP gene expression is 
put under control of Rous Sarcoma Virus (RSV)-LTR which does not respond 
to TAT. The results are set forth in FIG. I which shows that Compounds II 
is a specific inhibitor of TAT and does not exhibit nonspecific 
cytotoxicity. 
EXAMPLE 2 
Testing Compound II for Inhibition of HIV-Cytopathic Effect and Inhibition 
of Viral Antigen Production 
The assay protocols for the inhibition of HIV-cytopathic effect and the 
reduction of cell-associated viral antigens were modified from published 
procedures [Mitsuya, et al. P.N.A.S., USA 83:1911 (1986), and Hedenskog, 
et al., J. Med. Virol 19:325 (1986)]. 
High titer virus stocks (HIV-1 NIT strain) were grown in CD4.sup.+ CR10 
cells in RMPI-1640 media (Gibco Laboratories) supplemented with 10% fetal 
calf serum and 0.1 mg/ml Gentamicin. The collected media were filtered 
through a 0.8 Micron filter and virus isolates were concentrated 100 fold 
and stored at -80.degree. C. 
CD4.sup.+ CEM cells, propagated in the same medium, were incubated for 60 
minutes at 37.degree. C with diluted stock virus at MOI=1. Cells were 
washed three times with phosphate buffered saline and resuspended in the 
medium at 2.times.10.sup.5 cells/ml. Various quantities of Compound II 
were added. Four days after infection, numbers of live cells were counted 
by trypan blue exclusion [Mitsuya, et al. Proc. Natl. Acad. Sci. 83:1911 
(1986)]. At the same time, aliquots of cells were fixed with acetone and 
stained with antibodies from AIDS patients, followed by a second staining 
with fluorescein-conjugated goat anti-human IgG (Cappel). Cells stained 
with the fluorescent antibody were counted using a fluorescence microscope 
and the results were expressed as percentage of the total number of cells 
counted [(Hedenskog et al., J. Med. Virol. 19:325 (1986)]. For 
cytotoxicity testing, uninfected CEM cells were treated with Compound II 
at similar concentrations and toxicity of the compounds was measured by 
the live-cell count. Results of the assays are shown in FIG. II. 
EXAMPLE 3 
Formulations for Compound II 
______________________________________ 
TABLET FORMULATION I 
Item Ingredients Mg/Tablet 
______________________________________ 
1 Active ingredient 
20 mg* 
2 Starch 40 mg 
3 Avicel 80 mg 
4 Lactose 274 mg 
5 Magnesium Stearate 
2 mg 
416 mg 
______________________________________ 
Method for Preparation: 
1. Mix Items 3 and 4 in a suitable blender. 
2. Add and mix the drug to the mixture from Step 1. 
3. Add and mix Item 2 to the mixture from Step 2. 
4. Add and mix Item 5 to the mixture from Step 3. 
5. Compress the granulation on a suitable tablet press. 
______________________________________ 
TABLET FORMULATION II 
Item Ingredients mg/Tablet 
______________________________________ 
1 Active Ingredient 20 mg* 
2 Lactose 180 mg 
3 Pregelatinized Starch 
15 mg 
______________________________________ 
*The amount of active ingredient i.e. Compound I or Compound II may be 
varied as required. 
Method for Preparation: 
1. Mix Items 1, 2, 3, and 4 and granulate with water. 
2. Dry the granulation at 45.degree.-50.degree. C. 
3. Pass the granulation through a suitable mill. 
4. Add Items 5 and 6; mix. 
5. Compress the granulation on a suitable tablet press. 
______________________________________ 
SOFT GELATIN CAPSULE FORMULATION 
Item Ingredients mg/Tablet 
______________________________________ 
1 Active ingredient 20 mg* 
2 Ethoxylated Fatty acids 
500 mg 
3 PEG 4000 100 mg 
4 Vegetable Oils q.s. to 
1.0 g 
______________________________________ 
Method for Preparation: 
1. Add and mix drug with Items 2 and 4. 
2. Add Item 3 to the material from Step 1 and mix. 
3. Add vegetable oil to the required amount. 
4. Fill into a suitable capsule. 
______________________________________ 
ORAL LIQUID FORMULATION 
Item Ingredients mg/Tablet 
______________________________________ 
1 Active ingredient 
20.0 mg* 
2 Methylparaben 20.0 mg** 
3 Sucrose q.s. 
4 Flavoring Agent q.s. 
5 Citrate Buffer q.s. *** 
6 Purified Water q.s. 
5.0 mL 
______________________________________ 
*The amount of active ingredient may be varied as required. 
Method for Preparation: 
1. Dissolve Items 2, 4, and 5 into purified water. 
2. Add drug and dissolve into the solution from Step 1. 
3. Add Item 3 and dissolve. 
4. Add water to the required amount. 
5. Fill the solution into a suitable container. 
EXAMPLE 6 
Formulations for Compound II and ddC 
TABLET FORMULATION I 
______________________________________ 
TABLET FORMULATION I 
Item Ingredients Mg/Tablet 
______________________________________ 
1 Compound II 20 mg* 
2 ddC 5 mg 
3 Starch 40 mg 
4 Avicel 269 mg 
5 Lactose 269 mg 
6 Magnesium Stearate 
2 mg 
416 mg 
______________________________________ 
Method for Preparation: 
1. Mix Items 4 and 5 in a suitable blender. 
2. Add and mix Item 1 and 2 to the mixture from Step 1. 
3. Add and mix Item 3 to the mixture from Step 2. 
4. Add and mix Item 6 to the mixture from Step 3. 
5. Compress the granulation on a suitable tablet press. 
______________________________________ 
TABLET FORMULATION II 
Item Ingredients mg/Tablet 
______________________________________ 
1 Compound II 20 mg* 
2 ddC 5 mg** 
3 Lactose 175 mg 
4 Pregelatinized Starch 
15 mg 
5 Microcrystalline Cellulose 
72 mg 
6 Modified Starch 10 mg 
7 Magnesium Stearate 3 mg 
300 mg 
______________________________________ 
Method for Preparation: 
1. Mix Items 1, 2, 3, and 4 then granulate with water. 
2. Dry the granulation at 45.degree.-50.degree. C. 
3. Pass the granulation through a suitable mill. 
4. Add Items 6 and 7; mix. 
5. Compress the granulation on a suitable tablet press. 
______________________________________ 
SOFT GELATIN CAPSULE FORMULATION 
Item Ingredients mg/Tablet 
______________________________________ 
1 Compound II 20 mg* 
2 ddC 5 mg 
3 Ethoxylated Fatty acids 
500 mg 
4 PEG 4000 100 mg 
5 Vegetable Oils q.s. to 
1.0 g 
______________________________________ 
Method for Preparation: 
1. Combine and mix Items 1, 2, 3, and 5. 
2. Add Item 4 to the material from Step 1 and mix. 
3. Add vegetable oil to the required amount. 
4. Fill into a suitable capsule. 
______________________________________ 
ORAL LIQUID FORMULATION 
Item Ingredients mg/Tablet 
______________________________________ 
1 Compound II 4.0 mg* 
2 ddC 1.0 mg** 
3 Methylparaben 2.0 mg 
4 Propylparaben 0.2 mg 
5 Sucrose 100.0 q.s. 
6 Flavoring Agent q.s. 
7 Citrate Buffer 5.0 mg 
8 Purified Water q.s. 
1.0 mL 
______________________________________ 
Method for Preparation: 
1. Dissolve Items 3, 4, 6, and 7 into purified water. 
2. Add Items 1 and 2, and dissolve into the solution from Step 1. 
3. Add Item 5 and dissolve. 
4. Add water to the required amount. 
5. Fill the solution into a suitable container. 
______________________________________ 
ENTERAL FORMULATION 
Item Ingredients Mg/Tablet 
______________________________________ 
1 Compound II 20.0 mg* 
2 ddC 5.0 mg 
3 Propylene Glycol** 20.0 mg 
4 Emulphor 2.0 mg 
5 Water for Injection q.s. 
1.0 mg 
______________________________________ 
*The amount of Compound II may be varied as required. 
**The amount of ddC may be varied as required. 
***Solvents or solubilizers such as polyethylene glycols, alcohol, 
dimethylacetamide, glycerine, povidone, lecithin, sorbitan monooleate and 
trioleate, polysorbate 20 or 80 may be used in combination or alone to 
achieve the adequate solubility and stabilization. 
Method for Preparation: 
1. Dissolve Items 2 and 3 in water for injection. 
2. Add and dissolve the drug in the solution from Step 1. 
3. Adjust the pH using dilute sodium hydroxide or hydrochloric acid. NOTE: 
Buffers such as citrate, acetate, or phosphate may be incorporated for 
adequate stabilization. 
4. Add water for injection to the required amount. 
5. Fill the solution into a suitable contained.