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The roots of modern oncology: from discovery of new antitumor anthracyclines to their clinical use

Abstract

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 Streptomyces, found near Castel del Monte (Apulia), and to the skilled and motivated participants of both institutions, a new natural antitumor drug, daunomycin, was ready for clinical trials within 3 years. Patent interference by the Farmitalia French partner was overcome by the good quality of the Italian drug and by the cooperation between Prof. Di Marco, director of the Istituto Ricerche Farmitalia Research Laboratories for Microbiology and Chemotherapy, and Prof. Karnofsky, head of the Sloan-Kettering Cancer Institute of New York, leading to the first transatlantic clinical trials. The search for daunomycin’s sister anthracyclines led to the discovery and development of adriamycin, one of the best drugs born in Milan. This was the second act prologue of the history of Italian antitumor discovery and clinical oncology, which started in July 1969 when Prof. Di Marco sent Prof. Bonadonna the first vials of adriamycin (doxorubicin) to be tested in clinical trials. This article reviews the Milan scene in the 1960s, a city admired and noted for the outstanding scientific achievements of its private and public institutions in drugs and industrial product discovery.

Tumori 2016; 102(3): 226 - 235

Article Type: REVIEW

DOI:10.5301/tj.5000507

Authors

Giuseppe Cassinelli

Article History

Disclosures

Financial support: No financial support was received for this submission.
Conflict of interest: None of the authors has conflict of interest with this submission.

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Introduction

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 Claviceps paspalis and Claviceps purpurea (2, 3). Another Nobel Prize Laureate for Medicine, Prof. D. Bovet, awarded in 1957 for his studies concerning the mechanism of action of sulfamides, antihistaminics, and synthetic curares, directed the ISS laboratories of Therapeutic Chemistry in Rome from 1947 to 1964.

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 Table I, together with their suppliers, discovery and approval dates (4), along with their availability in Italy.

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).
Progesterone Lederle 1934 1939 Yes
Testosterone Legere 1935 1939 Yes
Dietylstilbestrol Lilly 1938 1944 No
Ethynylestradiol Schering 1938 1943 Yes
Mechloretamine Merck Sharp & Dhome 1946 1949 No
Methotrexate Lederle 1949 1953 Yes
6-Mercaptopurine Burroughs-Welcome 1932 1953 Yes
Chlorambucil Burroughs-Welcome 1953 1957 Yes
Busulfan Burroughs-Welcome 1953 1954 Yes
Melphalan Burroughs-Welcome 1954 1964 Yes
Prednisone Schein 1955 1965 Yes
Fluoximesterone Upjohn 1956 1956 No
5-Fluorouracil Roche 1957 1962 Yes
Cyclophosphamide Mead Johnson 1957 1959 Yes
Vinblastine Lilly 1958 1961 Yes
Uracyl Mustard Upjohn 1958 1962 No
Idoxyuridine Roussel-Uclaf 1959 1963 No
Dromostanolone Lilly 1959 1961 No
Vincristine Lilly 1961 1963 Yes

To receive approval by the National Cancer Institute (NCI), the drugs should have demonstrated and provided the following:

In vitro and in vivo inhibiting effect upon experimental tumors

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 Streptomyces genus, the main source of new antibacterial and antifungal agents for pharma contractors, but only a few of them were tested with the aim to identify antitumor agents. Of note, in a single year, 1962, 5,200 plant extracts were screened, along with 14,000 cultures of microorganisms in the fermentation program (8). Primary in vivo tests to screen synthetic agents and plant extracts were based on the evaluation of lifespan increase of mice bearing tumors, such as the Ehrlich carcinoma and Sarcoma 180, both in solid and ascite forms, the Yoshida 180 ascitic hepatoma, and the Walker carcinoma 256. Otherwise, in the fermentation program, contractors adopted in vitro prescreen cytotoxicity tests against human nasopharynx epidermoid carcinoma (9KB) and murine P388 leukemia cells, the last also used for in vivo tests (8).

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; in vitro screening for the identification of antibacterial, antifungal, and antiprotozoal cultures; and in vivo screening of microbial antitumor agents starting from 1957. Isolation and structural determination enabled the identification and production of new agents in amounts required for in vivo activity and toxicology studies in well-equipped animal houses. The proximity and rationality of the organization allowed, in a reasonable time, the full preclinical development required, at that time, for a potential new drug from a soil-isolated microorganism or from the chemist’s bench. Specific research programs could also benefit from collaborations of eminent scientists, such as the expert on Streptomyces taxonomy Prof. E. Baldacci working at the University of Milan and Pavia, and the eminent pharmacologist of the University of Pavia and Rome Prof. V. Erspamer, who discovered serotonin (enteramine) from marine organisms and biologically active peptides from the skin of South American frogs (9, 10).

Screening of antitumor agents from Streptomyces cultures at the FI research laboratories from 1957

At that time, the Streptomyces genus of the Actinomycetaceae family (now Actinobacteriaceae) was considered a good source of antibacterial agents and not of antitumor agents. For example, actinomycin D, isolated as antibacterial agent from Streptomyces antibioticus in 1940, was shown to display antitumor activity only in 1960 (11, 12). The choice of screening Streptomyces strains at FI was mainly based on other practical reasons, such as in-house expertise in their isolation, classification, and subemerged cultures, and their prevalence in soil. The antitumor screening program carried out in 1957 was mainly based on testing in vivo extracts of submerged cultures of newly isolated Streptomyces for their inhibitory activity against Ehrlich carcinoma and Sarcoma 180 in solid and ascitic form. Water-soluble fractions and preparations were also assayed in vitro for cytotoxicity on the human carcinoma Hela cell line. In 1959, the first active extract (13) obtained from cultures of the Streptomyces strain FI 1683, isolated from a soil sample collected in India, was found to contain compounds closely related to the anthracycline rhodomycin complex (14), the main component being likely identified as rhodomycin II or B (15). Further development of the extracts from Streptomyces strain FI 1683 as well as of the related anthracyclines cinerubin A and B from Streptomyces spp (16) was prevented by high toxicity in mice and rats. The early anthracyclines and related aglycones (anthracyclinones) were isolated mainly from mycelia of cultured Streptomyces species and structurally characterized by the group of Prof. H. Brockmann of the University of Göttingen, Germany (17).

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 Streptomyces strain coded as FI 1762. Some of the crude butanol extracts of both cultural broths and mycelia of the Streptomyces FI 1762, obtained from the pilot plant (headed by Dr. R. Barchielli and Dr. G. D’Amico) in December 1958 and February 1959 (crude C322), showed a promising antitumor activity against Ehrlich ascitic tumors in mice other than in vitro antibacterial and antifungal activity. Later in 1959, in the new antibiotic laboratory, under the responsibly of Dr. P.G. Orezzi, jointly with the newcomer Dr. G. Cassinelli, the skilled technicians G. Agosti, C. Corti, B. Pellegatta, and other coworkers started to search for the active components. Chromatographic analysis of extracts of the newer cultures of the Streptomyces FI 1762 revealed remarkable variability of the metabolite production, so that the researchers tried to correlate the culture components of the 7 isolated phenotypes to specific in vitro activities. Based on red metabolite higher production and cytotoxicity, the priority was given to type 1, and by morphologic, cultural, biochemical, and taxonomic studies, Dr. A. Grein, Dr. C. Spalla, and G. Canevazzi could identify the Streptomyces FI 1762 as a new species (18, 19). In May 1960, submitting the crude C322 to a separation by a countercurrent distribution apparatus, a red water-soluble fraction (1762-X-106-A) showing cytotoxic and antibacterial activity was obtained. A further preparative paper chromatography allowed the isolation of the major component, coded as FI 1762-B-101, to confirm its biological activities as well as to acquire its spectroscopic characteristics. These findings represented a milestone and allowed researchers to address the search for a water-soluble red component of an anthracycline-like complex endowed with in vitro cytotoxic and antibacterial activity. Thus, it was possible to follow the developmental process simply by testing in vitro the activity against Bacillus subtilis and/or by spectrometric determination of the complex at 495 nm. Analysis of the components could be done by paper chromatography followed by bioautography on plates of Bacillus subtilis cultures to identify which of the red components showed antibacterial activity (Fig. 1). In the summer of 1960, under the guide of Dr. P. Pennella, G. Ferni, and L. Pavesi, pilot plant fermentation of the Streptomyces FI 1762 showed an increased production of the FI 1762-B-101 from 1 mg up to 50 mg/L, leading to 4 crude butches (C-594-C597) endowed with antitumor activity. Such butches were the sources of the first significant lots of pure FI 1762-B-101, each weighting a few hundred milligrams, mainly used for in vitro and in vivo studies. These preparations were obtained by buffered powdered dry cellulose column partitioning chromatography, which also revealed some hydrophilic congeners. Some lipophilic metabolites, present in the last stage of fermentation, identified later as the toxic baumycin complex (20), could be hydrolyzed to FI 1762-B-101 by mild acidification, which inactivated toxic metabolites and facilitated its exit from mycelia and the culture filtration. The following solvent extraction and repartition steps allowed researchers to obtain crude FI 1762-B-101 (21).

Bioautography on a Bacillus subtilis plate of Whatman no. 1 paper chromatography, buffered at pH 5.4, developed for 16 hours at room temperature, with propanol:ethylacetate:water (7:1:2 v/v). B101 = daunomycin; B106 = adriamycin; B107 = 4’-daunosaminyl-daunomycin; B108 = likely 4’daunosaminyl-adriamycin.

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 vitro and in vivo studies of pharmacological and antitumor activities as well as to file the patent application of a novel identified microorganism producing a new antitumor agent renamed Streptomyces peucetius and daunomycin, respectively. Such names referred to the Peucetii and Daunii populations living in Apulia during the Bronze Age. Practically, the patent application was ready to be presented in April 1962, but the FI Patent Office raised some concerns about the novelty of daunomycin, because its structure was not yet completely elucidated. After an updated literature survey (difficult in the pre-Internet era), lack of authentic samples of the anthracyclines disclosed at that time such as pyrromycin and γ-rhodomycin complex (17) prompted Prof. Di Marco to send a sample of daunomycin to Prof. Brockmann to have his opinion about its novelty. Nevertheless, further data on the outstanding antitumor activity quickened the patent application filing on November 16, 1962 (inventors: A. Di Marco, G. Canevazzi, A Grein, P.G. Orezzi, M Gaetani; FARMITALIA Italian Patent Specification No. 29060). Two weeks later, the response of Prof. Brockman incorrectly associated daunomycin with the γ-rhodomycin complex (17). Increased production and availability of daunomycin allowed the researchers to demonstrate the broad spectrum of the antitumor activity on experimental tumors and to implement pharmacological and toxicological studies coordinated by Prof. Di Marco, both in FI and INT, and to make the intravenous (IV) formulation of pure daunomycin hydrochloride ready for human use. In July 1963, Prof. Di Marco provided Prof. Karnofsky at SKCI and MH with IV formulated vials for preclinical and clinical studies. In late 1963, other vials were sent to INT and other Italian centers mainly specialized for curing childhood leukemia. Preliminary data on antitumor activity and in vitro mechanism of action of daunomycin damaging both resting and dividing normal and tumor cells were reported (22). Taking advantage of daunomycin’s intrinsic fluorescence, it was possible to relate cytotoxicity to strong chromosomal aberrations and nuclear damage as well as to decrease in nucleolar size (23). An important report presented by Prof. Di Marco at the III International Congress of Chemotherapy (July 22-27, 1963) in Stuttgart, Germany (24), was followed by a seminal paper on February 15, 1964 (25), both showing the efficacy of daunomycin in experimental tumor models. In particular, the second report presented further data on the mechanism of action, evidencing that daunomycin could strongly bind to DNA, interfering with its functions also as a primer in RNA synthesis and in mitosis (25).

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 Streptomyces peucetius. Of note, 13-dihydro daunomycin was identified as the main metabolite of daunomycin in some animal species and humans (28).

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 Figure 1. Soon after, a simpler and more efficient method was adopted to assess the purity of B-101 and B-106 by a thin layer chromatographic analysis (TLC) on cellulose plates, buffered at pH 5.4, as shown in Figure 2. In the following 25 years, about other 20 new anthracyclines were isolated from cultures or obtained by biotransformation using mutant strains of Streptomyces peucetius (29). The TLC analytical method could easily evidence contamination by FI 1762-B-111 (daunorubicinol, NSC 180510) that, when tested by the NCI (30), appeared to be less active than daunomycin and 4 times more toxic (FI file, lethal dose 50 = 6.5 mg/kg, IV, in mice). The first pure sample of FI 1762-B-106 was sent to Prof. Di Marco in late 1963, followed by a more consistent sample of 200 mg in March 1964. Spectroscopic and elementary analysis along with chromatographic comparison of acid hydrolysis products with those of daunomycin showed for FI 1762-B-106 the same amino sugar and a more hydrophilic aglycone.

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 Streptomyces coeruleorubidus, filed on May 18, 1962, 6 months before that of daunomycin, and named rubidomycin in a report (31). Early in 1964, on the basis of spectral and chromatographic behavior, the main component of daunomycin and rubidomycin reference standard samples appeared indistinguishable (21). Thus, the structure determination of daunomycin became imperative. Owing to the effort of a team formed by Dr. W. Barbieri, Dr. G. Franceschi, and Dr. R. Mondelli (nuclear magnetic resonance [NMR] expert of the Polytechnic of Milan) joining Dr. P.G. Orezzi and Dr. G. Cassinelli, all headed by Prof. F. Arcamone, the structure determination of their components was achieved in late 1963 (Fig. 3). The peculiarity and novelty of the structure were identified in a methoxy group (OCH3) at 4-position, and acetyl (COCH3) side chain at position 9, both in the aglycone (daunomycinone) as well as in the new aminosugar (daunosamine). These features differentiating daunomycin from all known anthracyclines were considered essential for the antitumor activity, as later confirmed by structure-activity relationship studies on several other anthracyclines and derivatives. The structures of the daunomycin components were reported in August 1964 (32, 33) and the total absolute configuration, shown in Figure 3, in 1968 (34). As a consequence of the RP patent application priority, a reduced production of daunomycin was programmed for 1964 to support only the ongoing clinical trials in the United States and in Italy. The next year, as a result of a stock exchange, FI was fully acquired by Montecatini, and Rhodiatoce, an industry producing polymeric fibers (previously 49% of Montecatini and 51% of RP), by RP.

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 (Tab. I) (4), the top medical/oncologic literature was available, including the seminal papers on clinical classification of therapeutic responses to anticancer drugs authored by Prof. Karnofsky (36), those on the preclinical screening and evaluation of chemotherapeutic agents (37), and those on the nitrogen mustard mechanism of action (38).

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 Table I, the majority of the FDA-approved drugs that were available in Italy at that time were still used in 2015 (40).

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 Actinomyces coeruleorubidus in 1966, but named rubomycin C (43). In 1965, clinical trials went on at MH, and preclinical studies, coordinated by Prof. Di Marco, at SKCI (44) and INT, as reported in 1967 (45). In 1966, clinical reports of treatments of acute childhood leukemia at MH with daunomycin alone or in combination with prednisone were published (46). In the same year, preliminary clinical trials were also carried out in some Italian medical centers, such as the Pediatric Clinic of the G. Gaslini Hospital at Genoa (47).

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 Corriere della Sera on October 30 and published the same year (48). With reference to some of the most favorable cases of complete remission and disease-free survival after treatments including daunomycin, in 1965-1966 an interesting case report was described by De Castro et al (49). As a personal anecdote, in May 1975, while attending the annual American Chemical Society meeting in Philadelphia, I had the opportunity to watch on television, among the participants at an NCI fundraising program, 2 young people expressing their gratitude for having been cured of acute leukemia with “donomaisin” and for having been disease-free for more than 10 years.

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 Streptomyces peucetius, later named variant caesius. Incidentally, in 1966, Montecatini was incorporated into Montedison.

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 (Fig. 3), being titled “Adriamycin derivatives.” The corresponding US patent no. 3,590,028 was issued on September 26, 1971. The breakthrough discoveries of the new anthracyclines are reported in Figure 4. Table II illustrates, in more detail, the dates of the discovery and patent of rubidomycin (RP, FI) and doxorubicin (FI) and their US approval dates, with adriamycin the winner. Its name (after being 1762 B-106 or hydroxydaunomycin) referred to the good view of the Adriatic Sea from Castel del Monte, homeland of the parental producing strain. In April 1967, the FI researchers went back to Settimo T at the “Posticcio Steroidi,” which in the meantime had been restructured. The daunomycin production scaling-up and the reasonable carcinogenic activity of anthracyclines had prompted the FI management to realize a safe environmental pilot plant, providing workers with safety protective equipment and adequate decontamination and containment systems.

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.

Acknowledgement

The author thanks his daughter Giuliana Cassinelli for help in organization and preparation of the manuscript, suggestions, and discussion.

Disclosures

Financial support: No financial support was received for this submission.
Conflict of interest: None of the authors has conflict of interest with this submission.
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Authors

Affiliations

  • Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Milan - Italy

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