In May 1960, the Farmitalia CEO Dr. Bertini and the director of the Istituto Nazionale dei Tumori of Milan Prof. Bucalossi (talent scout and city’s Mayor) signed a research agreement for the discovery and development up to clinical trials of new natural antitumor agents. This agreement can be considered as a pioneering and fruitful example of a translational discovery program with relevant transatlantic connections. Owing to an eclectic
Tumori 2016; 102(3): 226 - 235
Article Type: REVIEW
- • Accepted on 11/03/2016
- • Available online on 21/04/2016
- • Published in print on 02/06/2016
This article is available as full text PDF.
The aim of this article is to provide an overview of a program of discovery and development of new biosynthetic antitumor agents involving the Istituto Ricerche Farmitalia (FI) from 1957 and the Istituto Nazionale dei Tumori (INT) from 1960, both located in Milan. The collaboration agreement between the 2 institutes was signed in May 1960 by Dr. Giulio Bertini, the FI Managing Director, and Prof. Pietro Bucalossi, Director of INT. By the terms of the agreement, a new unit of clinical chemotherapy was to be established within INT. Moreover, researchers, some paid by FI, were to become involved in INT laboratories equipped for pharmacologic and preclinical studies of new agents discovered in the FI Research Laboratories for Microbiology and Chemotherapy, directed by Prof. Aurelio Di Marco. He supervised all the programs up to 1964, when he officially became the director of the Division of Experimental Oncology B, a position he held until 1979 in INT, maintaining his collaboration with FI up to his death in 1984. In a couple of years, the division of Experimental Oncology and the Department of Chemotherapy of the INT were organized similarly to the Sloan-Kettering Cancer Institute (SKCI) and the Memorial Hospital (MH) of New York, with the advantage of being only 6 kilometers from the FI discovery laboratories. Financial support from both partners, the pioneering guidance of Prof. Di Marco, and osmosis of skilled and motivated researchers allowed them to realize the basis of an agile and fruitful program for discovering new antitumor agents. Three years after the FI-INT agreement signature, a new antitumor drug (daunomycin), obtained from a microbial culture, was ready to be used for clinical trials, with the authorization of the Italian Health Ministry, in accordance with the current regulations. The results of the collaboration between FI and INT in the 1960s are well-documented by several books, reviews, and reports (cited later). This article reports some undisclosed episodes and original FI laboratory records of the 1960s, through the author’s personal recollection and documentation, in a Milan to remember for its scientific successes.
The early 1960s: the golden quinquennium of the Italian scientific discoveries, collaborating with three Nobel Prize Laureates
The early 1960s were years of prosperity and development for the Italian economy and industry. Owing to private and public investments of the previous decade, coincident with the so-called Italian boom, outstanding scientific results were achieved in the golden quinquennium. In 1961-1962, Prof. P. Sensi, director of the Lepetit Department of Natural Product Research of Milan, and his collaborators discovered rifamycin SV (Rifampin), a very effective drug still used to cure tuberculosis (1). In 1963, the Nobel Prize for Chemistry was co-assigned to Prof. G. Natta for the discovery of isotactic polypropylene realized in the laboratories of the Polytechnic of Milan with the support of the chemical industry Montecatini. This holding, the main owner of FI, promoted an avant-garde type of multidisciplinary research program, nowadays named translational, involving academic and public research laboratories. Another of these research programs, concerning the discovery and development of natural ergolines (i.e., a class of pharmacological active alkaloids), between FI and the Istituto Superiore di Sanità (ISS) led some FI researchers to work in Rome in the ISS International Center for Chemical Microbiology, directed by Prof. E.B. Chain, the Nobel Prize Laureate for Medicine in 1945 for his studies of penicillin structure and production. Microbiological and chemical studies in the ISS and FI laboratories realized a methodology for producing lysergic acid, ergotamine, and derivatives in submerged cultures of fungal strains of
1964: Discovery in the United States and approved anticancer drugs in the United States and Italy
To clarify the context in which the anthracyclines were introduced, it is necessary to consider the antitumor agents available in the 1960s. Those present in the United States in 1964 are reported in
Discovery and FDA-approved anticancer drugs in the United States in 1964a and their availability in Italy in 1964b and in 2015c
|Name||Supplier||Discovery||FDA approval||Presence in Italy in 1964 and 2015|
|FDA = Food and Drug Administration.|
|a Adapted from reference 4.|
|b The US-approved drugs in 1964 also available in Italy in 1964 (39) and c 2015 (40).|
|Mechloretamine||Merck Sharp & Dhome||1946||1949||No|
To receive approval by the National Cancer Institute (NCI), the drugs should have demonstrated and provided the following:
pharmacological studies in animals concerning the mechanism of action, mode of administration, and toxicity
Through a preliminary phase of clinical trials, qualified as clinical pharmacology, the mode of administration and the maximum tolerated dose in a sufficient number of patients
A minimum of 2 independent and well-controlled clinical trials, demonstrating, usually as monotherapy, cures or probable cure, and favorable effects on survival and quality of life in some of the patients
These outlines were developed by the Food and Drug Administration (FDA) following The FDA and Cosmetic Act of 1938 and The Drug Amendment of 1962 for the requirements to assure safety, effectiveness, and reliability of drugs (4).
At that time, the first drugs, discovered through animal experiments, were androgens and estrogens, approved in the early 1940s for the treatment of breast and prostate carcinomas. At the same time, synthetic agents such as the nitrogen mustard mechlorethamine (5) and the folic acid antagonist aminopterin were found able to induce some remissions in malignant lymphomas and pediatric acute leukemia, respectively (6). In the 1950s, a great number of synthetic agents were prescreened. The most relevant centers focusing on anticancer drug development were the SKCI in the United States, earlier directed by Prof. C.P. Rhodes and then by Prof. D. Karnofsky, the Chester Beatty Research Institute in the United Kingdom, and the University of Tokyo in Japan. The US NCI, established in 1936 and mainly interested in searching for antitumor agents in plant extracts, was appointed by the US Congress to coordinate the first National Cooperative Cancer Chemotherapy Program with the associated SKCI in 1955. Such a program was sponsored by the NCI, FDA, American Cancer Society, and other institutions. The original aim was to acquire and screen drugs and compounds other than plant extracts, to perform, by contracts, preclinical studies and clinical trials of active agents through special grants. In the next 3 years, the NCI program was fed by a large voluntary submission of products from academic and industrial sources. In 1957, the natural source program included also fermentation products mainly provided by contractors, the US pharmaceutical companies Upjohn, Parke-Davis, and Bristol-Myers, and the Institute of Microbial Chemistry of Tokyo (7). After 4 years spent evaluating fungal cultures, stimulated by wartime industrial penicillin production, from about 1961 the program was focused on cultures of the
Discovery and preclinical development of new agents in the FI Research Laboratories
The FI Research Laboratories of Milan were confined in an area of a hectare in 1- or 2-floor buildings, enclosing 3 courtyards, where in 1955 about 200 people were employed in the well-equipped chemical research laboratories, directed by Prof. B. Camerino, and microbiology and chemotherapy, directed by Prof. A. Di Marco. The 2 units were supported by common facilities, such as chemical and physical analyses, pilot plants, pharmaceutical technology, and a radionuclide laboratory. The microbiology and chemotherapy division, starting from a soil sample, could perform microbial isolation and taxonomic classification, cultures, and scaling of up to fermenters of 500 liters; extract preparation;
Screening of antitumor agents from
Streptomyces cultures at the FI research laboratories from 1957
At that time, the
The multiform and eclectic
Streptomyces FI 1762
In the summer of 1957, a work outing to Apulia was organized for hundreds of FI employers, headed by the Scientific Director Dr. G. Bertini. A researcher, G. Canevazzi, collected a soil sample near the famous Castel del Monte at Andria, built in the XIII century, during the reign of Swabian Emperor Frederick II. From that soil sample, she isolated a
Bioautography on a
In February 1962, a fermentation provided 3 grams of pure and crystalline product. This allowed researchers to set up extraction and purification procedures and physicochemical characterization and to complete
In July 1963, preliminary structural studies of daunomycin (21) showed that mild acid hydrolysis gave a lipophilic aglycone (daunomycinone) and a hydrophilic aminosugar. Comparison with authentic samples of β-rhodomycinone (13), ε-pyrromycinone (26), and rutilantinone (27) showed the peculiarity of the daunomycinone structural features, ruling out the assignment to γ-rhodomycin complex.
Analysis of daunomycin co-metabolites, isolation and preliminary characterization of the new congener FI 1762-B-106
Increased fermentation volumes and daunomycin production mainly in the FI industrial plant of Settimo T near Turin allowed the isolation and characterization of some coproduced metabolites present in the head (more lipophilic) and tail (more hydrophilic) fractions of the daunomycin chromatographic purification columns. Later on, some metabolites were identified, such as the following:
Daunomycin and adriamycin dates of application up to approval
|FI code||1st Italian name||Patent||Approval|
|Italian application||USA issue||INN||Italy||USA|
|FI = Istituto Ricerche Farmitalia; INN = international nonproprietary name.|
|a Rubidomycin (RP) US patent issued in 1976.|
|b Wyet, Ives RP Licensee.|
|1762 B-101||Daunomicina||Nov 1962||1977 (FI)||Daunorubicina||Daunoblastina June 1968||Daunorubicin 1979b|
|1762 B-106||Adriamicina||Mar 1967||1971||Doxorubicin||Adriblastina Dec 1971||Adriamycin Aug 1974|
1762-B-104, likely 4’-O-vancosaminyl daunomycin (IMI 107)
1762-B-105, as baunomycin-like (20)
1762-B-106, more hydrophilic but less stable than B101 (adriamycin)
1792-B-107, as 4’-O-daunosaminyl daunomycin
1762-B-111, as 13-dihydro daunomycin (daunorubicinol)
Both 4’-O-daunosaminyl daunomycin and 13-dihydro daunomycin were described in US patent 3,683,163 (August 22, 1972) as metabolites of a mutant strain of
In September 1963, from a pool of chromatographic tail fractions, 50 mg of pure FI 1762-B-106 was obtained by Dr. Cassinelli as hydrochloride. The bioautography of the paper chromatography of daunomycin (B-101), pure B-106, and congeners B-107 and B-111 are shown in
Thin layer chromatography (TLC) analysis of daunomycin congeners on a TLC cellulose plate (DC-Fertigplatten-Merck), buffered at pH 5.4. From the left side: lane 1: FI 1762-B-107: 4’-O-daunosaminyl-daunomycin; lane 2: FI 1762-B106: adriamycin; lane 3: FI 1762-B111: 13 dihydro-daunomycin; lane 4: FI 1762-B101: daunomycin; lane 5: crude daunomycin; lane 6: FI 1762-B104: likely 4’vancosaminyl-daunomycin; lane 7: FI 1762-B102: daunomycinone; lane 8: mixture 1 to 7 (5 µg each); lane 9: mixture 1 to 7 (2.5 µg each).
1964: Structure determination of daunomycin and French patent application interference
In December 1963, FI researchers became aware of the publication by Rhône-Poulenc (RP) (French owner of 49% of FI) of the patent application of a compound very similar to daunomycin (13,057 RP) from
Daunomycin and adriamycin structure.
Memorial Sloan-Kettering Cancer Institute of New York: A fundamental crossroad of Italian oncologic chemotherapy
In the 1960s, besides being the site of frequent, friendly, and fruitful contacts between Prof. Karnofsky and Prof. Di Marco, the MH and SKCI had the opportunity to welcome in 1962 Dr. Gianni Bonadonna as recipient of a scholarship at the Clinical Chemotherapy Division, later working directly with Prof. Karnofsky (35). At the SKCI and MH, together with all the FDA-approved anticancer drugs (
In the same period, few pharmacokinetics studies in experimental animals were reported, mainly carried out using radioactive labeled [3H, 14C] agents, expensive, and difficult to prepare and to manage in special protected radionuclide laboratories and animal houses. The lack of other sensitive and practical analytical methods as well as instruments to study pharmacokinetics in animals, and, more importantly, in patients, made oncologic chemotherapy a difficult and somewhat empirical mission at that time.
In September 1964, the director of INT and newly elected Mayor of Milan, Prof. P. Bucalossi, met the young oncologist Dr. Gianni Bonadonna in New York and convinced him to come back to Milan as a head of the INT Clinical Chemotherapy Unit (reparto C) (39), to update chemotherapeutic agents and regimens. As shown in
Early clinical trials of daunomycin
An initial clinical trial was carried out at INT by Dr. S. Di Pietro in 1964 (41) on 20 patients with different types of cancer, 16 receiving the drug IV and 4 by intra-arterial route. The dose ranged from 5 to 20 mg/day and the total from 25 to 400 mg, the highest total dose being about 8 mg/kg. Two patients developed phlebitis in the injection site, and leukopenia without thrombocytopenia was generally observed at the high drug doses. Improvement was observed in patients with lymphocytic leukemia and reticulum cell sarcoma and melanoma following intra-arterial drug administration. Other clinical trials were carried out in Italy mainly on patients with childhood leukemia. Likely due to the low doses, there was no adequate response and consequently, no further interest was shown by clinicians. Thus, FI researchers tried to get daunomycin vials back. About 150 vials were collected in refrigerators at FI, and Dr. Curcio at the medical direction of FI was informed. Soon after, on June 2, 1964, on urgent request from Prof. D. Karnofsky, the vials reached the United States. Within a couple of months, local US media spread news regarding complete or partial remissions of acute leukemia in children receiving daunomycin. This news reinforced the trilateral collaboration (SKCI-FI-INT) and the production of daunomycin in the second semester of 1964, and attenuated the anxiety of the RP priority at FI. The choice of treating leukemias was based on the activity of daunomycin on 2 experimental murine leukemia models, L1210 and P388, both considered, at that time, of great predictive value. The first results of the clinical studies on childhood leukemia were published by Tan et al in 1965 (42).
Regarding rubidomycin, the first clinical trials started at the Hospital Saint Louis in Paris in 1965. The promising results of the first clinical trials of daunomycin made the search for other anthracyclines a hot topic. Indeed, soon after, on the other side of the Iron Curtain, rubidomycin was reisolated from
A survey summarizing the clinical data of all the daunomycin-treated cases at MH, with particular reference to childhood leukemia, was presented by C. Tan at the 9th International Congress on Cancer held in Tokyo on October 23-29, 1967, as first reported by Prof. Bucalossi in the Italian newspaper
During his frequent visits at FI, Prof. Di Marco was always asking for 1762 B-106, which showed better activity on several experimental tumors in comparison with daunomycin. Taking advantage of the daunomycin increased production in the 1,000-L fermenter plant of Settimo T, and requests for analytical support, with the technician C. Pol, Dr. Cassinelli spent some time in the summer of 1966 at the “Posticcio Steroidi,” a building used for the final purification step of daunomycin on cellulose columns. From a crude preparation, the third lot of 1762-B-106 (170 mg) was obtained. Part of this was used for structural preliminary analysis, along with the first samples obtained by the submerged cultures of the strain FI 106 mutant
1967: A year for daunomycin rise and adriamycin debut
On March 11, 1967, a presentation of preclinical and clinical data of daunomycin and rubidomycin obtained in the United States and in Europe was held at the Saint Louis Hospital of Paris (50). The efficacy demonstrated in these and other domestic clinical trials allowed the approval of Cerubidine (rubidomycin RP) in France and Daunoblastina (daunomycin FI) in Italy in 1968.
In 1967, the 2 companies RP and FI in joint communications announced that rubidomycin and daunomycin had been demonstrated to be the same product (51, 52) and from 1969, the generic name daunorubicin was adopted by WHO. In 1967, in view of increasing number and size of the daunomycin clinical trials granted in the United States, NCI set up a contract with the Stanford Research Institute (Menlo Park, CA, USA) to compare commercial samples from FI and RP. The response (53) confirmed the identity of the main component, but reported “The commercial samples showed similar, but definitely not identical spectra (infrared, ultraviolet and NMR) … By thin-layer chromatography, commercial daunomycin was shown to be homogeneous and commercial rubidomycin was shown to be heterogeneous …”
On July 1, 1967, NCI set up a purchase contract with FI (through Chemor Corp., the FI representative for the United States) of 1 kilogram of daunomycin hydrochloride, 800 grams formulated for IV injection and 200 grams as bulk, to be delivered by June 1, 1968.
In the meantime, chemical physical characterization and antitumor activity data of FI 1762-B-106 completed the Italian application patent filed on April 18, 1967, titled “An antibiotic B106 FI produced by the mutant strain FI 106 (Inventors: F. Arcamone, G. Cassinelli, A. Di Marco, M. Gaetani).” A year later, the corresponding US patent application (filed on April 18, 1968) first disclosed the name and structure of adriamycin (
Timeline of new anthracycline discovery.
By the end of 1967, an additional gram of 1762-B-106 was obtained, partially used to complete its structural determination as 14-hydroxy daunomycin (54, 55). In February 1968, colleagues of Settimo T sent 5 grams of pure adriamycin, essential to implement preclinical and formulation studies, to INT and FI, respectively. At that time, the major concerns were the shortage of drug and the current recovery (few mg/L) referred to the volumes of both mutant strain submerged cultures and daunomycin fermentators, where the ratio was 1 to 100. It became urgent to find an alternative way to obtain adriamycin. Owing to the chemical knowledge acquired in daunomycin structure determination (56), a team consisting of Dr. G. Franceschi, Dr. S. Penco, and S. Redaelli, headed by Prof. F. Arcamone, set up an efficient laboratory procedure to obtain adriamycin from the easily available daunomycin (Italian patent application no. 15159 A/68, April 12, 1968) (57). By this approach, a first lot (1 gram) in early 1968 and a second one (50 grams) in May of pure adriamycin hydrochloride were obtained (58).
The higher availability of adriamycin as well as additional data obtained by Prof. Di Marco and coworkers on the outstanding activity of the drug against experimental tumors speeded up preclinical, pharmacologic, toxicologic, and formulation studies. The results were presented in Milan at the International Symposium on Adriamycin, September 9-11, 1971 (59). At the end of the first act, Prof. Di Marco as the deux ex machina raised the curtain on the second act, sending IV formulated adriamycin vials to Prof. Gianni Bonadonna at INT. He described the event in the foreword of a volume updating in 1984 the epirubicin studies and dedicated to Prof. Di Marco, who died on January 4, 1984 (60). The text is as follows: “At the beginning of July 1968, a young laboratory technician stepped in my office carrying a carton box containing 30 vials of adriamycin (doxorubicin) as well as a brief report which summarized the chemical properties and the preclinical results of this daunorubicin analogue. ‘A gift from Doctor Di Marco—you may wish to test it in patients,’ she said. I asked for some additional details and I spent the rest of the month discussing with my coworkers the plans for a Phase I study. In September, after returning from vacation, we began to treat our first patient.”
This event represents the beginning of the second act, showing the important role of Prof. Bonadonna in the rise and development of clinical oncology not only in Italy, but worldwide.
The author thanks his daughter Giuliana Cassinelli for help in organization and preparation of the manuscript, suggestions, and discussion.