Results of a double-blind placebo-controlled study of the double-stranded RNA drug polyI:polyC12U in the treatment of HIV infection.

In this multicenter, randomized, double-blind study the activity of polyI:polyC12U administered with zidovudine was evaluated in the treatment of HIV infection. Thirty-six HIV-positive, pre-AIDS individuals (100-500 CD4+ cells/mm3) who had had at least six months of zidovudine therapy received polyI:polyC12U (400 or 700 mg) or placebo twice weekly with zidovudine. PolyI:polyC12U subjects with baseline CD4+ counts > or = 300/mm3 showed a trend towards reduced CD4+ loss versus placebo recipients. PolyI:polyC12U subjects were more likely to exhibit positive delayed-type hypersensitivity responses than placebo recipients. Placebo subjects crossing over to polyI:polyC12U therapy demonstrated improved CD4+ and delayed-type hypersensitivity responses. PolyI:polyC12U subjects with baseline CD4+ counts > or = 300/mm3 were less likely to develop AIDS than similar placebo subjects. PolyI:polyC12U therapy of HIV-positive subjects restored or stabilized immune function as indexed by delayed-type hypersensitivity reactivity and, in individuals with CD4+ counts > 300/mm3, abrogated CD4+ loss and reduced disease progression. PolyI:polyC12U was generally well-tolerated in this zidovudine-treated population. No subject discontinued therapy due to an adverse reaction or aberrant laboratory parameter.

Ampligen inhibits human herpesvirus-6 in vitro.

The recently discovered human herpesvirus-6 (HHV-6) is being associated with an increasing number of conditions in which there is evidence of immunologic dysfunction. A number of widely available antiviral agents have shown little or no activity against the virus. We found that Ampligen [Poly (1): Poly (C12U), a synthetic, mismatched, double-stranded RNA, has potent, previously unexpected antiviral effects. Cells known to allow replication of HHV-6 were infected with the virus and treated with Ampligen under various conditions. When cells were pretreated with Ampligen (concentrations of 100 or 200 micrograms/ml) prior to infection or treated shortly after infection, viral replication was inhibited by 46-98%. At 100 and 200 micrograms/ml, Ampligen also inhibited the DNA polymerase activity of HHV-6 by 42-98%. When lower concentrations of Ampligen (10 and 50 micrograms/ml) were used, only pretreatment of cells, with Ampligen, followed by virus infection and carrying the infected cells with Ampligen, significantly inhibited HHV-6 infection (83.7 and 89.1% respectively). Indirect evidence suggests that Ampligen may inhibit viral attachment to cellular receptors and/or inhibit intracellular maturation of the virus. The above concentrations of Ampligen were not toxic to the cells used in the study. Given these in vitro findings, and the low frequency of toxicity reported with the use of Ampligen, clinical trials of this drug in patients with evidence of reactivated HHV-6 infection would seem to be warranted.

Synergistic inhibition of AZT-resistant HIV by AZT combined with poly(I):poly(C12U), without synergistic toxicity to bone marrow progenitor cell elements.

Mutation of human immunodeficiency virus (HIV) to drug resistance is an obstacle to HIV containment, and may account for the transitory nature of the improvement in CD4 cell counts of patients receiving azidothymidine (AZT). The emergence of AZT-resistant (AZTR) virus might be suppressed if a second therapeutic could be added; however, such a regimen would have to confer not only additional control over HIV replication but also no additional toxicity, especially to bone marrow progenitor cells. In the present study, HIV was isolated from patients receiving AZT alone and was studied for sensitivity to the mismatched double-stranded RNA, poly(I):poly(C12U) (ampligen). In addition, the combination of poly(I):poly(C12U) plus AZT was studied in vitro for toxicity to bone marrow CFU-GM and in patients receiving combined therapy for bone marrow toxicity. HIV isolated from patients receiving AZT alone showed higher resistance to AZT than wildtype virus, but remained sensitive to poly(I):poly(C12U). Poly(I):poly(C12U) and AZT were synergistic in inhibiting all isolates of HIV tested, regardless of their AZTR phenotype. Furthermore, the combination of poly(I):poly(C12U) and AZT showed no toxicity in vitro to bone marrow CFU-GM compared to AZT alone. In 11 HIV infected individuals receiving the combinational regimen, bone marrow function gradually improved. These results indicate that poly(I):poly(C12U) was active against AZTR HIV, synergistic with AZT and did not convey added toxicity.

A controlled clinical trial with a specifically configured RNA drug, poly(I).poly(C12U), in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a physically debilitating illness associated with immunologic abnormalities, viral reactivation, and impairment of cognition. In a randomized, multicenter, placebo-controlled, double-blind study of 92 patients meeting the CFS case definition of the Centers for Disease Control and Prevention, the response of several laboratory and clinical variables to an antiviral and immunomodulatory drug, poly(I).poly(C12U), was determined. Measures of clinical response included Karnofsky performance score, a cognition scale derived from a self-administered instrument assessing symptomatology (SCL-90-R), an activities of daily living scale, and exercise treadmill performance. After 24 weeks, patients receiving poly(I).poly(C12U) had higher scores for both global performance and perceived cognition than did patients receiving placebo. In particular, patients given poly(I).poly(C12U) had increased Karnofsky performance scores (P < .03), exhibited a greater ability to do work during exercise treadmill testing (P = .01), displayed an enhanced capacity to perform the activities of daily living (P < .04), had a reduced cognitive deficit (P = .05), and required less use of other medications (P < .05).

Antitumor effects of interleukin-2 and mismatched double-stranded RNA, individually and in combination, against a human malignant melanoma xenograft.

The antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000-20,000 units) of rIL-2 and mismatched dsRNA (500 micrograms). The in vivo tumor growth was significantly inhibited by the combined treatments (P less than 0.05) and survival was significantly increased (P less than 0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer (NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P less than 0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P less than 0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.

Mismatched double-stranded RNA, Ampligen (poly(I): poly(C12U), demonstrates antiviral and immunostimulatory activities in HIV disease.

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

Differential effects of human natural interferon-alpha and mismatched double-stranded RNA against a human renal cell carcinoma xenograft.

The antitumor effects of natural human IFN-alpha and mismatched dsRNA against the human renal cell carcinoma cell line 786-0 were studied both in a clonogenic soft agar assay and in the nude mouse. The 786-0 cells were sensitive in vitro to the antiproliferative effects of IFN-alpha in a dose-response manner, up to 3000 IRU/ml. These cells were also sensitive, in a dose-dependent manner, to mismatched dsRNA in the clonogenic assay. Mismatched dsRNA was effective in inhibiting tumor growth (p less than 0.001) in nude mouse xenografts, with regression of the tumor mass seen in all animals. A significant increase in survival (p less than 0.001) was seen in the mismatched dsRNA treated group. In contrast, IFN-alpha did not inhibit tumor growth in vivo, even though significant titers of IFN-alpha (greater than 3,000 IRU/ml) were found in the serum shortly after treatment. Mismatched dsRNA did not induce the production of human IFNs by the tumor cells in vitro. Assays of mouse IFN induction and their in vitro antigrowth effects indicated that the in vivo antiproliferative effect of mismatched dsRNA was probably not due to potentiation of any direct effects by the induced mouse IFNs. Tumor growth inhibition appeared to occur, at least in part, from the significant augmentation (p less than 0.01) of natural killer cell activity by mismatched dsRNA, as measured in the spleen cells of treated mice. These results suggest that, although both IFN-alpha and mismatched dsRNA can be directly antiproliferative against this tumor, either the IFN-independent antitumor effects of mismatched dsRNA or the mismatched dsRNA-induced augmentation of the host immune response plays a major role in tumor regression. Potentially, both mechanisms may be important in this system.

Detection of poly(I):poly(C12U), mismatched double-stranded RNA, by rapid solution hybridization: blood values after intravenous infusion.

A rapid solution hybridization technique has been developed for estimating blood concentrations of poly(I):poly(C12U), a high molecular weight bioactive double-stranded RNA. Samples were prepared by mixing 100 microL of blood with, 165 microL of 6 M guanidine thiocyanate (GuSCN) and 0.16 M EDTA pH 8.0, and freezing. Hybridizations were carried out with a [3H]poly(C) probe in 3 M GuSCN for 10 min at 37 degrees C. Ribonuclease-resistant hybrids were collected by precipitation with trichloroacetic acid and filtration. Validation studies demonstrated minor interassay variance; the assay was accurate in the range 0.1 to 10 ng poly(I):poly(C12U). Thirty-one blood samples from 15 patients were collected and prepared before and immediately after an average 35 min intravenous infusion of 40-500 mg poly(I):poly(C12U). Postadministration values averaged 48% (s.d. 23%) of the theoretical maximum (range 20-102%). These results confirm previous observations of rapid elimination kinetics of poly(I):poly(C12U) in patients.

Augmented antitumor effect of combined human natural interferon-alpha and mismatched double-stranded RNA treatment against a human malignant melanoma xenograft.

The antitumor effect of combined natural human interferon-alpha (IFN) and mismatched double-stranded RNA (dsRNA) treatment against the human malignant melanoma cell line, BRO, was studied. In vitro results, using a tissue culture antiproliferative assay, indicated that these cells were moderately sensitive to IFN-alpha. In contrast, mismatched dsRNA had no antitumor effect, and a minimal stimulation of cell growth, over part of the concentration range tested, was observed. Mismatched dsRNA did not potentiate the antitumor effect of IFN-alpha in cells receiving combination treatment. Xenografts of BRO cells, inoculated subcutaneously into nude mice, were used to evaluate the antitumor effects of IFN-alpha and mismatched dsRNA. Growth of the primary tumor was inhibited by both drugs alone or in combination (p less than 0.001), but the combined treatment was most effective and appeared to be additive. The number of spontaneous lung metastases was also inhibited (p less than 0.02) in all treatment groups. Survival, however, was significantly increased only in the IFN-alpha/mismatched dsRNA group (p less than 0.02 compared to controls, p less than 0.05 compared to mismatched dsRNA alone). Determination of splenic natural killer (NK) cell activity against BRO cells demonstrated that significantly augmented NK activity to the same extent, but that the IFN-alpha alone had no effect. These results indicate that IFN-alpha worked through direct antiproliferative mechanisms while mismatched dsRNA stimulated host immunomodulatory effects. The increased tumor growth inhibition and survival in the dual treatment group appears to result from the combined direct antiproliferative and indirect immunomodulatory effects.

Growth of astrocytomas in the human tumor clonogenic assay and sensitivity to mismatched dsRNA and interferons.

Nine astrocytoma specimens were received from seven patients and processed for testing in the human tumor clonogenic assay (HTCA). Cells derived from these specimens were challenged with human natural alpha-interferon (alpha-IFN) and beta interferon (beta-IFN), recombinant beta interferon (beta ser-IFN), and mismatched double-stranded (ds) RNA (Ampligen). Six of the astrocytoma specimens formed adequate colonies for drug sensitivity testing (greater than or equal to 30 colonies/plate), and all were high-grade (III-IV) tumors. Sensitivity was defined as a greater than or equal to 50% decrease in tumor colony formation following drug exposure and was observed with alpha-IFN (2/4), beta-IFN (3/4), and mismatched dsRNA (4/5) exposure. No decrease in colony growth was observed after recombinant beta ser-IFN exposure, and in 2 of 3 cases, colony formation was stimulated. The sensitivity of 75 non-CNS solid tumors to mismatched dsRNA was compared to the high-grade astrocytomas in the HTCA. Of the 10 additional histologic tumor types studied, carcinoid and renal cell carcinomas exhibited the greatest sensitivity to mismatched dsRNA: 63% and 52%, respectively. However, in comparison, 80% of the high-grade astrocytomas were sensitive, demonstrating that these gliomas are among the most sensitive of human tumors to mismatched dsRNA in vitro. Clinical trials of interferons and mismatched dsRNA, coupled with in vitro sensitivity studies, should further define their therapeutic potential.

Clinical, immunological, and virological effects of ampligen, a mismatched double-stranded RNA, in patients with AIDS or AIDS-related complex.

10 patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), or lymphadenopathy syndrome (LAS) were given 200-250 mg ampligen, a mismatched double-stranded (ds) RNA with in-vitro antiviral activity against human immunodeficiency virus (HIV), twice a week for up to 18 weeks, without side-effects or toxicity. In all 9 patients who were positive for HIV RNA in peripheral blood mononuclear cells before therapy, levels became undetectable between days 10 and 40 of the start of therapy. 6 of the 7 patients with ARC or LAS also showed a progressive reduction in HIV load as measured by co-culture assays. All 10 patients had augmentation of delayed-type hypersensitivity skin reactions. Other changes noted during ampligen therapy included an increase in or maintenance of numbers of helper-inducer T lymphocytes, improvements in HIV-related symptoms, rises in titre of neutralising antibodies against HIV, and restoration of proper functioning of the natural lymphocyte antiviral dsRNA-dependent (2'-5'-oligoadenylate/RNA-ase L) pathway. Thus, in the short term, ampligen seems to have the dual ability to restore immunological function and to control HIV replication.

Implications of retroviral and oncogene activity in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells (a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neoplasia in general.

Mismatched double-stranded RNA (ampligen) reduces concentration of zidovudine (azidothymidine) required for in-vitro inhibition of human immunodeficiency virus.

'Ampligen', a non-toxic, mismatched polymer of double-stranded RNA with antiviral and immunomodulatory activities reduced the concentration of zidovudine (azidothymidine, AZT; 'Retrovir', Wellcome) required for inhibitory activity against human immunodeficiency virus (HIV) in vitro. At the higher doses of AZT tested, the virustatic activity observed seemed to have a synergistic virustatic relation with ampligen. Thus, combined therapy with ampligen and AZT can be expected to be more beneficial than AZT alone to patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex since AZT regimens that seem to be clinically effective are associated with considerable toxicity.

Antiproliferative effect of mismatched double-stranded RNA on fresh human tumor cells analyzed in a clonogenic assay.

Colony growth in soft agar was used to identify human tumors that were sensitive to the direct antiproliferative effects of mismatched dsRNA (Ampligen). The results suggest that different human solid malignancies vary significantly in their sensitivity to Ampligen. Tumors with 50% or more of their surgical specimens showing sensitivity included carcinoid, glioblastoma, and carcinomas of the kidney, and lung. Resistant tumors (less than 15% sensitivity) included sarcomas and colo-rectal carcinomas. Overall, 42% of the tumor specimens studied showed a 50% or greater reduction in tumor cell colony formation after a single initial treatment with Ampligen (250 micrograms/ml). Interestingly, one patient's tumor which was de novo sensitive to interferon (IFN), but emerged as IFN-resistant following IFN therapy, remained sensitive to Ampligen. Thus, a clonogenic assay may prove useful in identifying human tumors and individuals for clinical trials with Ampligen, including patients resistant to IFN.

Familial occurrence of breast cancer is associated with reduced natural killer cytotoxicity.

A factor in the incidence of spontaneous neoplasms in mice is the endogenous level of natural cell-mediated cytotoxicity. These immunosurveillant or host defense mechanisms are probably under the control of multiple gene products including interferons. We studied natural killer (NK) cytotoxicity using peripheral blood mononuclear cells from 59 normal individuals with either a high (17) or low (42) familial incidence of breast cancer. The K562 cell line was used as target in 51Cr release assays. Three effector: target ratios (6.2:1, 25:1, and 50:1) were studied in quadruplicate using 3, 4 and 5-h incubations. Significantly lower natural killer activity (p less than 0.002) was detected in normal individuals with high familial incidences of breast cancer compared to individuals with low incidences in each of the three separate assays (50:1). The same conclusion was reached whether the data were expressed in terms of lytic units per 10(7) blood mononuclear cells or as % specific 51Cr released. Thus, a relationship was observed between the occurrence of breast cancer in closely related family members and low natural cell-mediated cytotoxicity. This result suggests that defects in NK activity may play a role in the initiation of human breast tumors. However, prospective studies will be necessary to establish whether low NK cell activity is a risk factor for breast cancer.

Comparative studies of ampligen (mismatched double-stranded RNA) and interferons.

Historically, it has been assumed that double-stranded (ds) RNAs function at a cellular level exclusively via an interferon (IFN) induction mechanism. However, current studies conducted both in the laboratory and at the clinical level reveal that this assumption is incorrect and, indeed, underestimates the intrinsic antitumor activity of certain dsRNAs. A specific dsRNA (Ampligen) shows strong antiproliferative activity against human carcinoid tumor cells in a clonogenic assay when natural alpha- and beta-IFNs were inactive. Similarly, in vivo studies in which human renal cancer cells were transplanted into athymic mice demonstrate a strong antitumor effect of Ampligen whereas such tumors are largely unaffected by alpha-IFN treatment. In a comparative study including many fresh human tumors of various histological types (breast, ovarian, melanoma, renal, and carcinoid) numerous examples were uncovered of Ampligen sensitivity (antiproliferative effect) in the face of relative or complete insensitivity to IFNs. Synergistic effects of Ampligen plus IFN overcame the resistance of some human tumor cells to either biological modifier given alone. It can also be demonstrated that the antitumor action of Ampligen on certain human lung tumor cells is not shared by polyinosinic . polycytidylic acid, thus indicating that different dsRNAs may themselves exhibit dissimilar effects on various human tumors.

Clinical studies with ampligen (mismatched double-stranded RNA).

Results of an ongoing clinical study of a mismatched double-stranded (ds) RNA, termed Ampligen, in patients with metastatic cancer are described. In a pilot study of Ampligen (lot 1) involving mostly hematologic malignancies, patients received cumulative doses up to approximately 450 mg without untoward effects. Evidence of biologic/antitumor effects was observed (3/5 patients) by monitoring tumor-specific markers or tumor cell morphology. In patients with solid tumors receiving lot 2, Ampligen cumulative doses over 4 g were well tolerated. The drug was given by intravenous infusion (10-80 mg/infusion, twice weekly), in some instances for more than 1 year, without clinically significant side effects. Specifically, no evidence of hematologic, liver, or renal toxicity, which was previously noted with other dsRNAs, was observed. Side effects consisted of occasional mild fatigue or flu-like symptoms. Fever, when encountered, was transient and low grade (less than 100.5 degrees F). Importantly, an analysis of patient sera for dsRNA antibodies revealed that no patient had evidence of specific antibodies directed against Ampligen. Other dsRNAs cause up to a 60% incidence of antibody formation. Additionally, a novel method was developed to monitor Ampligen blood levels. In a survey of seven patients, Ampligen had a mean plasma half-life of 23 min. Ampligen administration can also result in activation of both natural killer (NK) cells and a lymphocyte, interferon-associated, intracellular enzyme. Dose-dependent antitumor effects were seen in several solid tumors; in doses of 10-40 mg, 3/9 patients showed stable disease for up to 1 year. At the 80-mg dose level, 2/5 patients showed tumor regressions (mixed and partial responses).(ABSTRACT TRUNCATED AT 250 WORDS)