Abstract:
This invention relates to a method of treating basal cell carcinoma with purified human recombinant DNA interferon alpha-2, preferably purified human recombinant DNA interferon alpha-2b by administering intralesionally (by injection) to a patient in need of such treatment, a sufficient amount of the purified human recombinant DNA alpha interferon, preferably recombinant DNA interferon alpha-2b, to be effective as an antitumor agent.

Description:
RELATED APPLICATIONS 
     This is a continuation-in-part of U.S. patent application Ser. No. 866,644, filed May 27, 1986, now abandoned, the priority of which is claimed. 
    
    
     BACKGROUND 
     This invention relates to a method of treating basal cell carcinoma with recombinant human alpha interferon by administering the interferon directly into the carcinoma lesion, i.e. intralesionally. 
     Basal cell carcinomas are the most common cutaneous neoplasms found in humans. The majority of the 500,000 new cases of nonmelanoma skin cancers each year are basal cell carcinomas. 
     Basal cell carcinomas exist in a variety of clinical and histological forms such as nodular-ulcerative, superficial, pigmented, morphealike, fibroepithelioma and nevoid syndrome. Present treatment methods include various surgical techniques such as electrodesiccation and curettage, excision, cryosurgery and irradiation. Cure rates for the surgical techniques are generally stated to be about 95%. Despite the high cure rates effected by surgical techniques, non-surgical methods of therapy are generally thought to be more desirable. 
     Various efforts have been made to treat cancers by injecting interferon directly into the lesion. For example, Ikeda, Gan to Kagaku Ryoho, 12(4), 936-942 (1985) used recombinant interferon A to treat various malignant skin tumors and achieved low cure rates. None of the tumors treated were stated to be basal cell carcinomas. Sikora, K. (Editor), Interferon and Cancer, Ikic, D., Intralesional Therapy, 169-181, Plenum, N.Y. (1983) used unpurified human leukocyte interferon to treat basal cell carcinoma patients intralesionally. Ikic did not use a purified interferon material, but used a material containing a mixture of leukocyte interferon components and non-interferon impurities. 
     Barenbein, et al., Vestn. Dermatol. Venerol. No. (4): 31-33 (1985) used a &#34;new Soviet interferon preparation, human leukocyte interferon for injection II&#34; to treat skin basal cell carcinoma. It is not clear from the article if the &#34;new&#34; interferon is a recombinant human alpha interferon. The duration of the course of treatment varied from 10 to 20 days with a maximum dose of 100,000 International Units. The patients were injected twice daily for ten days, in a dose of 2,500-5,000 International Units, until a total dose of 50,000-100,000 IU was reached. This dosage is different than required to achieve the results of the present invention. Ikic et al., The Lancet, May 9, 1981, pp. 1025-1027, discloses local administration of crude leukocyte interferon to cancer patients, including those with basal cell carcinoma. Langer et al., Journal of Investigative Dermatology, 83, 128s-136s (1984) discloses purification, bacterial expression and biological activities of human interferons. Langer et al. state &#34;Subsequent work has demonstrated that there are at least 10 highly related human alpha interferons (IFN-α) each with characteristic chemical and biological properties and each encoded by a different gene&#34;. In a discussion of leukocyte interferons, Langer et al. on page 130s stated: 
      &#34;The purification of naturally induced human leukocyte interferons conclusively established several concepts; (1) Multiple leukocyte interferon species can be induced simultaneously in cultured human cells. This immediately suggested the existence of multiple genes corresponding to each of these structurally distinct species. (2) These interferons are closely related, having similar, but not identical molecular weights, amino acid content, and cryptic maps. (3) Although all the species were active on both human and bovine cells, the relative specific activities, particularly on human cells, differed considerably. (4) No carbohydrate was detected on five of the purified species examined. This contradicted the previously accepted notion that all interferons were glycoproteins.&#34; 
     and noted that the leukocyte interferons had different activities: &#34;It was found that all the species exhibited antiproliferative activity on these cells [human lymphoblastoid Daudi cell line], although their potencies differed. This supported the notion that the chemical differences of the leukocyte interferon species are reflected in functional differences.&#34; 
     Langer et al. further observed that: 
      &#34;If the various effects of interferon--antiviral, antiproliferative, natural killer cell activation, etc.--were all mediated through the same biochemical pathway, then it might be expected that the potency of a species in one assay would correlate with its potency in another assay. However, this was not observed.&#34; 
     There is no discussion in Langer et al. regarding treatment of basal cell carcinoma. Staehelin et al., Methods in Enzymology, Pestka ed., 78, 505,511(1981), published prior to Langer et al. supra, discusses a homogeneous human leukocyte prepared from bacterial fermentations. The authors state: &#34;The recombinant IFLrA exhibits antiviral activity and antiproliferative activity comparable to crude and purified natural leukocyte interferons.&#34; The identity of the species of interferon is not apparent, so it cannot be ascertained which of the numerous leukocyte interferon species is being discussed. There is no discussion of treatment of basal cell carcinoma. 
     Weissman, U.S. Pat. No. 4,530,901 discloses a means to make leukocyte type interferons with recombinant DNA molecules. 
     Interferons are a family of proteins which exhibit antiviral activity against certain viruses and anticancer activity against certain cancers. There are three types of interferons; alpha or leukocyte interferon, beta or fibroblast interferon and gamma or immune interferon. Human alpha interferon is a naturally occurring mixture of at least eleven components including those designated alpha-1 interferon and alpha-2 interferon. Human alpha interferon exhibiting biological properties similar to those of naturally occurring human leukocyte interferon can be made by recombinant methods. 
     A number of alpha interferon species or components are known and are usually designated by a numeral after the Greek letter alpha, and all are contemplated for use in this invention. Thus, the species designated human alpha-1 interferon and human alpha-2 interferon (sometimes called human alpha-2  interferon which includes human alpha-2a and human alpha-2b interferon; USAN: Interferon Alfa-2 including Interferon Alfa-2a and Interferon Alfa-2b) are contemplated, with human alpha-2 interferon preferred and Interferon Alfa-2b most preferred. Interferon alfa-2 can be produced in bacteria using recombinant techniques as disclosed in Rubenstein, Biochem. Biophys. Acta, 695, 5-6 (1982). In addition, interferon alfa-2 may be prepared by recombinant-DNA methods disclosed by Nagata et al., Nature, 284, 316-320 (1980), European Patent 32,134 and U.S. Pat. No. 4,289,690. Various alpha-2-interferon species are disclosed in U.S. Pat. No. 4,503,035. Preferred for use in this invention is purified human recombinant DNA interferon alfa-2b. 
     SUMMARY OF THE INVENTION 
     This invention relates to a method of treating basal cell carcinoma with purified human recombinant DNA interferon alpha-2preferably purified human recombinant DNA interferon alpha-2b by administering intralesionally (by injection) to a patient in need of such treatment, a sufficient amount of the purified human recombinant DNA alpha interferon, preferably purified recombinant DNA interferon alpha-2b, to be effective as an antitumor agent. 
     DETAILED DESCRIPTION 
     As used herein &#34;alpha interferon&#34; means purified human recombinant DNA interferon alpha-2 which includes purified human recombinant DNA interferon alpha-2a and purified human recombinant DNA interferon alpha-2b. In most instances this invention will be described in the following discussion using the preferred interferon as purified &#34;human recombinant DNA interferon alpha-2b&#34; or &#34;interferon alpha-2b&#34;. 
     For intralesional administration, liquid injectable pharmaceutically acceptable compositions are used. Such compositions can, for example, be prepared by diluting freeze dried purified human recombinant DNA interferon alpha-2b with sterile water with or without preservatives although one with preservatives is preferred, to produce an isotonic solution containing the appropriate concentration of the interferon. Other injectable compositions using saline, aqueous dextrose, glycerol, ethanol and the like, to thereby form a solution or suspension for injection can also be used. If desired, minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate, can be incorporated into the compositions. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; see for example, Remington&#39;s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The amount of interferon alpha-2b administered is critical only to the extent that it is effective for the therapeutic purpose. The quantity in the composition or formulation administered will, in any event, be an amount effective to achieve an anti-basal cell carcinoma effect in the subject being treated. 
     The amount of purified human recombinant DNA interferon alpha-2b in a 0.15 ml. injectable dosage is about 1.5×10 6  I.U. (International Units), however, if a range of dosages is required, such dosages can be made, e.g., vials can be made with 5×10 6  to 2.25×10 7  I.U. 
     Clinical tests to determine the effect of the purified interferon alpha-2b on basal cell carcinoma were conducted. In one test, interferon alpha-2b was administered in doses of 1.5×10 6  I.U. three days a week for three weeks, i.e. 13.5×10 6  I.U. total. In another test the purified interferon alpha-2b was administered with six injections of 2.25×10 6  I.U. over two weeks, i.e. 13.5×10 6  I.U. total. In another test, patients with basal cell carcinoma lesions of from 3-10 cm 2  received about 0.5×10 6  I.U./cm 2  of initial lesion area of purified interferon alpha-2b three times a week every other day for three weeks (9 doses), i.e. a total of from 13.5×10 6  I.U. to 45×10 6  I.U. per patient depending on the size of the lesion. Patients with basal cell carcinoma lesions of from 2-3 cm 2  may receive 1.5×10 6  I.U. of purified interferon alpha-2b three times a week for three weeks, i.e. a total of 13.5×10 6  I.U. units. 
     Although these doses and the regimen described were beneficial, it is contemplated that they be considered only guidelines and that the attending clinician will determine, in his or her judgment, an appropriate dosage and regimen, using the patient&#39;s age and condition as well as the severity of the basal cell carcinoma. 
     The following illustrates the effects of treating patients having basal cell carcinoma with intralesionally administered interferon alpha-2b. 
    
    
     CLINICAL STUDY I 
     PATIENTS AND METHODS 
     Patients 
     Eight patients, six males and two females, each with one biopsy proven primary basal cell carcinoma of the nodular (3 patients) or superficial (5 patients) type were included in the study. The lesions, which varied in size from 7×6 mm to 14×12 mm, were located on the back (5), shoulder (1), arm (1) and neck (1). The diagnosis of nodular or superficial basal cell carcinoma was confirmed by incisional biopsy at least one week prior to the initiation of treatment. 
     Each patient was in good health and elected to be treated with interferon alpha-2b rather than undergo other ablative or surgical procedures. 
     Laboratory Test 
     Laboratory tests, hematology, serum chemistries and urinalysis were performed prior to, during and following treatment. 
     Treatment 
     Treatments were conducted with freeze-dried purified human recombinant DNA alpha-2b interferon which was in vials. The interferon the vials was diluted with sterile, preservative-free water to produce an isotonic solution containing sufficient interferon concentration so that 0.15 ml of solution contained 1.5×10 6  International Units (IU). Each lesion was injected with 0.15 ml of interferon alpha-2b with a 30-gauge needle on a tuberculin syringe. The needle was inserted tangentially into the lesion with care being taken to inject the entire amount intralesionally. The procedure was repeated for a total of three injections per week for three weeks. Thus, each lesion was injected with a total of 13.5×10 6  I.U. 
     Patients were evaluated during the treatment for clinical response and side effects. Evaluations were continued at one, four and eight weeks after completion of treatment. Systemic side effects (i.e. flu-like symptoms) were treated and controlled with oral acetaminophen. 
     Response Criteria 
     Excisional biopsy was performed on the lesional test site of each of the eight patients eight weeks following completion of the treatment with interferon alpha-2b. Multiple serial sections were examined histopathologically after staining with hematoxylin and eosin. Clinical responses were measured during treatment and follow-up visits through evaluation of changes in lesion size, erythema and, in the case of nodular lesions, percentage of flattening. 
     The following Table I shows the results of treatment of basal cell carcinoma with purified recombinant DNA human interferon alpha-2b. 
     
                                           TABLE I__________________________________________________________________________Results of Treating Basal Cell Carcinomawith Interferon Alpha-2b.          Pre   Post  % Lesion    Type of          Treatment                Treatment                      Flattening                             Follow-up    Basal Cell          Lesion Size                Lesion Size                      (nodular                             ExcisionalCase   Age Sex    Carcinoma          (mm)  (mm)  lesions only)                             Biopsy__________________________________________________________________________1  59 M  superficial          8 × 8                6 × 3                      --     No tumor noted3  63 M  superficial          8 × 5                0     --     No tumor noted5  50 F  superficial          14 × 12                3 × 2                      --     No tumor noted7  51 M  superficial          11 × 8                6 × 4                      --     No tumor noted8  58 M  superficial          12 × 10                6 × 4                      --     No tumor noted2  52 M  nodular          7 × 6                5 × 4                      100    No tumor noted4  50 F  nodular          9 × 8                6 × 3                      100    No tumor noted6  48 M  nodular          11 × 6                6 × 4                      100    No tumor noted__________________________________________________________________________ 
    
     As is apparent from the data in Table I, all the basal cell carcinomas were eliminated. This was confirmed by histopathologic study. 
     The sites where the basal cell carcinomas had been treated demonstrated ectasia of vessels in the papillary dermis, perivascular lymphocytic and histiocytic accumulations, and in some cases lymphocyte exocytosis, civatte body formation and incontinence of pigment. No &#34;tumor&#34; stroma, necrotic basal cell carcinoma, polymorphonuclear neutrophil leukocytes (PMN), transformed lymphocytes or eosinophils were found. 
     SIDE EFFECTS 
     No life threatening or serious treatment related side effects were found as shown in the following Table II. 
     
                       TABLE II______________________________________Side Effects when Treating Basal Cell Carcinomawith Interferon alpha-2b.Patient #    1     2     3   4   5   6   7   8   Total______________________________________Fever (p first dose)        +     +     +   +   +           +   6Fever (p other doses)    +   +       +           3Malaise      +                   +   +   +   +   5Itching (at site)  +         +           +       3Lightheadedness          +                       1Pain (at site)               +                   1Muscle aches                 +               +   2Joint aches                  +           +       2Depressive mood              +                   1Headache                         +       +       2Abdominal discomfort             +               1Chills                                   +       1______________________________________ All of the above were mild or moderate, except some of the malaise symptoms were severe. 
    
     As the data in Table II indicate fever was the most common side effect followed by malaise. All side effects were transient and reversible, and all patients were able to complete the treatment regimen. 
     White blood cell counts were diminished in three patients. In each case the white blood cell count returned to normal before the program was completed. 
     The side effects observed were those to be expected with the use of intralesional administration of interferon. It was noted that the side effects were more frequent at the first injection and became fewer as the program progressed. 
     CLINICAL STUDY II 
     PATIENTS AND METHODS 
     Patients 
     One hundred sixty five patients, 29 females and 136 males, each with biopsy proven primary basal cell carcinoma (BCC) of nodular (89 patients) or superficial (76 patients) types were included in the study which was a randomized, double-blind, parallel group, multicenter study design. The lesions, which varied in size from 15 mm 2  to 400 mm 2  were located on the back (45), neck (20), shoulder (24), forehead (11), head and face (27), trunk (17) and extremities (21). 
     Each patient was in good health and elected to be treated with interferon alpha-2b rather than undergo other ablative or surgical procedures. 
     Laboratory Test and Treatment 
     The laboratory tests and treatment were the same as described for Clinical Study I except evaluations of the patients after completion of the treatment were continued at one, four, eight and twelve weeks after treatment. 
     Response Criteria 
     Punch biopsy was performed for histopathologic evaluation of the test site. 
     The following Tables show the results of treatment of basal cell carcinoma with purified recombinant DNA human interferon alpha-2b in Clinical Study II. 
     
                       TABLE III______________________________________Results of Treating Basal Cell Carcinoma with InterferonAlpha-2b (16-week biopsy).                     Percent      Punch Biopsy Results*                     of LesionsTreatment    N#      Negative   Positive                               Cured______________________________________Interferon    120     102        18      85Placebo   42      12        30      29TOTAL    162______________________________________ *Biopsy results: Negative = tumor absent, Positive = tumor present. #Biopsy data are missing for three Interferontreated patients. 
    
     As is apparent from the data in Table III, 85% of the treated basal cell carcinomas were eliminated, whereas only 29% of the placebo treated lesions tested negative in the punch biopsy. The punch biopsy was taken 16 weeks after the treatment began. 
     
                       TABLE IV______________________________________Results of Treating Basal Cell Carcinomawith Interferon Alpha 2b,by Lesion Type (16-week Biopsy)Type of      Punch Biopsy Results*BCC    Treatment N.sup. Negative                           Positive                                  % Cured______________________________________Super- Interferon            56     49       7     88ficial Placebo   19      6      13     32Nodular  Interferon            64     53      11     83  Placebo   23      6      17     26______________________________________ *Biopsy results: Negative = tumor absent, Positive = tumor present. .sup. Biopsy data are missing for three Interferontreated patients. 
    
     The results in Table IV demonstrate that efficacy of interferon alpha-2b is independent of lesion type. 
     
                       TABLE V______________________________________Results of Treating Basal Cell Carcinomawith Interferon Alpha-2b,by Lesion Size (16-week biopsy)BaselineTest Lesion   Punch Biopsy Results (Week 16)*Area (sq mm)     N.sup.  Negative  Positive % Cured______________________________________&lt;50       53      42        11       79&gt;50-100   35      33        2        94&gt;100-200  24      20        4        83&gt;200       8       7        1        88TOTAL     120______________________________________ *Biopsy results: Negative = tumor absent, Positive = tumor present. .sup. Biopsy data are missing for three patients. 
    
     The results in Table V show that the lesion size did not significantly affect the efficacy of the interferon. 
     It was found during the treatment period, the mean area of the test sites treated with interferon alpha-2b increased progressively, and by the 4-week visit (first week post treatment), had increased 64% above the area measured at baseline (pretreatment lesion size); this was in contrast to an insignificant change in area of placebo-treated lesions. At the 8-week visit, mean lesion area in the interferon treated lesions had decreased by 26% from baseline compared to a decrease of 11% in the placebo treatment group. The area of the interferon lesions progressively decreased and at the 16-week evaluation, the mean decrease in lesion area was 46% whereas the mean decrease in lesion area at the 16 week evaluation in the placebo treatment group was 16%, significantly less than the interferon treated group. The patients were followed for one year and the results indicated that the overall cure rate at the end of the year was 81%. 
     Side Effects 
     No life threatening or serious side effects were found. The side effects reported were almost always mild or moderate in severity and were similar to those reported in Clinical Study I. 
     The following are a description of a clinical study on the treatment of large basal cell carcinomas by intralesional administration cf purified human recombinant DNA interferon alpha-2b. 
     CLINICAL STUDY III 
     PATIENTS AND METHODS 
     Ten patients, all males, each with biopsy proven basal cell carcinoma lesions of nodular (5 patients) and superficial (5 patients) types were included in the study which was an open-label study. The mean lesion area at baseline (pretreatment measurement) was 6.1 cm 2  and ranged from 3.4 to 9.9 cm 2 . 
     The test lesions were located on the back (4 patients), shoulder (2 patients), neck (2 patients), arm (1 patient) and temple (1 patient). 
     Each patient was in good health and elected to be treated with interferon alpha-2b rather than undergo other ablative or surgical procedures. 
     Laboratory Test 
     Laboratory tests were performed at baseline during treatment and during follow-up. 
     Treatment 
     Treatments were conducted with freeze-dried purified human recombinant DNA interferon alpha-2b which was in vials containing 5×10 6  I.U. Each lesion of from about three to ten cm 2  in area was injected intralesionally with about 0.5×10 6  I.U./cm 2  of original lesion area. The procedure was repeated for a total of three injections per week for three weeks resulting in a total dosage in this procedure of 16 to 45×10 6  I.U. per patient, depending on the lesion size. 
     Patients were evaluated during the treatment for clinical response and side effects. Evaluations were made during the three week treatment period and for 13 weeks after the completion of treatment. Systemic side effects (i.e. flu-like symptoms were treated and controlled with oral acetaminophen. 
     The following Table VI show the results at the end of 16 weeks. 
     
                       TABLE VI______________________________________Results from End-of-Study(16 week) Biopsies (3 punch biopsies)                          Results.sup.3  Type                    Presence orPatient  of Lesion Baseline Area (cm.sup.2)                          Absence of Tumor______________________________________2      superficial            6.5           absent4      superficial            5.3           absent.sup. 5.sup.1  superficial            8.5           .sup. present.sup.26      superficial            9.5           absent10     superficial            6.5           absent1      nodular   3.4           absent3      nodular   4.5           absent7      nodular   3.5           absent8      nodular   3.8           absent9      nodular   9.9           present______________________________________ .sup.1 Lesion had nodular component. .sup.2 Two biopsies were negative (tumor absent), the third was positive (tumor present). .sup.3 Results of three biopsies per patient. 
    
     The results in Table VI show that treatment with interferon alpha-2b resulted in no tumors remaining in 80% of the patients. 
     The following Table VII shows the changes in lesion sizes as a result of treatment with interferon alpha-2b. 
     
                       TABLE VII______________________________________Effect of Interferon Alpha 2-bon Lesion SizeLesion Type    Lesion Area (cm.sup.2)Patient  Baseline  Posttreatment.sup.                           Percent Change______________________________________Superficial2        6.5       3.4          -484        5.3       3.5          -34 5       8.5       8.6           +16        9.5       0.1          -9910       6.5       2.0          -69Nodular1        3.4       2.0          -413        4.5       3.7          -187        3.5       0.3          -918        3.8       1.8          -53 9       9.9       10.0          +1MEAN     6.1       3.4          -45______________________________________ .sup. Week 16 evaluation.  Tumor present at end of study. 
    
     The data in Table VII show that all lesions had changes in lesion area. For those lesions that had negative biopsies at week 16, the measured area of the lesional site had decreased from 18% to 99%. In these biopsy negative lesions, the measurable area at Week 16 could be characterized as pigmentary and textural skin changes. At the last visit prior to biopsy, the lesion sites that were tumor free were variously described as slightly depressed or hypopigmented patches, pink in color, or mildly erythematous. Of the patients cured, six listed cosmetic results as excellent and two as very good. 
     The two patients (No. 5 and 9) with tumor present at Week 16 had slight increases in lesion area. For these patients, the lesions were described as a pearly pink nodule or nodular lesion. These descriptions indicate that the presence of tumor could be determined by clinical examination. 
     Side Effects 
     No life threatening or serious side effects were found. Those present were predominantly the expected flu-like symptoms which accompany treatment with interferon. These symptoms were most often reported during the first week of treatment, with fewer reports during weeks 2 and 3. Three patients reported mild local reactions such as pain, burning or itching. 
     Analyses indicated no detectable levels of circulating interferon neutralizing factors in any patient. 
     The results of the above described clinical testing programs show that intralesional injection of purified recombinant DNA human interferon alpha-2b is a safe, effective treatment for basal cell carcinoma.