The Mechanism of Action of Cyclophosphamide and Its Consequences for the Development of a New Generation of Oxazaphosphorine Cytostatics

Scientia Pharmaceutica

Review The Mechanism of Action of Cyclophosphamide and Its Consequences for the Development of a New Generation of Oxazaphosphorine Cytostatics

Georg Voelcker

Institute of Biochemistry II, Goethe University Frankfurt Medical School, 60590 Frankfurt, Germany; [email protected]; Tel.: +49-69-6301-5652; Fax: +49-69-6301-5577 Received: 7 August 2020; Accepted: 15 September 2020; Published: 28 September 2020

Abstract: Although cyclophosphamide (CP) has been used successfully in the clinic for over 50 years, it has so far not been possible to elucidate the mechanism of action and to use it for improvement. This was not possible because the basis of the mechanism of action of CP, which was found by lucky coincidence, is apoptosis, the discovery of which was honored with the Nobel Prize only in 2002. Another reason was that results from cell culture experiments were used to elucidate the mechanism of action, ignoring the fact that in vivo metabolism differs from in vitro conditions. In vitro, toxic acrolein is formed during the formation of the cytotoxic metabolite phosphoreamidemustard (PAM), whereas in vivo proapoptotic hydroxypropanal (HPA) is formed. The CP metabolites formed in sequence 4-hydroxycyclophosphamide (OHCP) are the main cause of toxicity, aldophosphamide (ALDO) is the pharmacologically active metabolite and HPA amplifies the cytotoxic apoptosis initiated by DNA alkylation by PAM. It is shown that toxicity is drastically reduced but anti-tumor activity strongly increased by the formation of ALDO bypassing OHCP. Furthermore, it is shown that the anti-tumor activity against advanced solid P388 tumors that grow on CD2F1 mice is increased by orders of magnitude if DNA damage caused by a modified PAM is poorly repairable.

Keywords: cyclophosphamide; thiazolidine and perhydrothiazine derivatives of aldophosphamide and I-aldophosphamide; anti-tumor activity; toxicity; mechanism of action

1. Introduction The development of cyclophosphamide (CP) was based on the idea of enzymatically converting a non-toxic transport form into the active form at the site of action. This idea was already realized in the drug “Honvan” (diethylstilbestrol diphosphate) used in the treatment of prostate cancer. Honvan is an ineffective prodrug that is split into the active form diethylstilbestrol in prostate tissue by acid phosphatases. This principle should be imitated when developing a cytostatic. The first and only chemotherapeutic agent available at that time was nitrogen mustard originally produced as chemical warfare agent. The aim was to convert the highly toxic nitrogen mustard into a non-toxic form by incorporating it into a phosphoric acid amide bond from which it should be released in the tumor tissue by phosphoamidase; it was believed at the time that the enzyme phosphoamidase, which is specific for catalyzing the hydrolysis of phosphorus-nitrogen bonds, was present in increased activity in the tumor tissue [1,2]. This concept turned out to be wrong because the assumption of increased phosphoamidase activity in tumor cells was not correct. However, the result of the substances synthesized according to this premise was CP one of the most successful chemotherapy drugs for tumor therapy. It was quickly recognized that CP is an inactive prodrug that is hydroxylated by cyt. P450 in the liver to 4-hydroxycyclophosphamide (OHCP). All attempts in the last 50 years to improve CP, which was

Sci. Pharm. 2020, 88, 42; doi:10.3390/scipharm88040042 www.mdpi.com/journal/scipharm Sci. Pharm. 2020, 88, 42 2 of 13 found by serendipity failed because the real mechanism of action of CP was unknown. An example of the failed attempt to improve oxazaphosphorine cytostatics (OX) is the development of Glufosfamide (β-D-glucose-isophosphoreamide mustard), which has been synthesized on the basis of the rationale that cancer cells have an increased uptake of glucose. Glufosfamide was developed with the intention of enriching the alkylating moiety of Ifosfamide—that is isophosphoreamide mustard—in tumor cells [3]. The hoped-for increase in eﬀectiveness did not materialize because the second component, the triggering of apoptosis by the OX metabolite 3-hydroxypropanal (HPA), was not taken into account due to ignorance of the true mechanism of action. Only after the discovery of apoptosis and the enzymatic cleavage of the CP metabolite aldophosphamide (ALDO) with the formation of the proapoptotic CP metabolite 3-hydroxypropanal (HPA) was it possible to develop a conclusive concept for the mechanism of action of CP. The aim of this review is to show how, in knowledge of the mechanism of action and the main causes of toxicity, a new generation of OX can be developed. In animal experiments, model compounds of this new OX are by orders of magnitude more eﬀective than the ones currently used in the clinic.

2. Summary on Materials and Methods For the synthesis of aldophosphamide-thiazolidine (N,N-2-(2-chloroethyl)-phosphoric acid-diamide-2-(20-thiazolidinyle-40-carbonic acid-)-ethylester, NSC-613060), see [4]. Aldophosphamide perhydrothiazine (N,N-2-(2-chloroethyl)-phosphoric acid-diamide-2-(20-10,30-thiazinyle-40-carbonic acid-)-ethylester was synthesized according to Dausmann [5]. The aldophosphamide-perhydrothiaznie derivatives IAP (N-(2-chloroethyl)-N0-(2-chloroethyl)-phosphoric acid-amide-2-(20-10,30 -thiazinyle-40-carbonic acid-)-ethylester, and SUM-IAP (N-(2-chloroethyl)-N0-(mesylethyl)-phosphoric acid-amide-2-(20-10,30-thiazinyle-40-carbonic acid-)-ethylester, were synthesized according to Zimmermann et al. [6]. 4-S-ethanol-sulfido-cyclophosphamide (N,N-2-(2-chloroethyl)-amino)-N-2 -oxo-4-ethanesulfido-1,3,2-oxazaphospho- rinane) was synthesized as described in [7]. Determination of CP and the CP metabolites OHCP, ALDO, 4-ketocyclophosphamide (KCP) and carboxyphosphamide (CARB) was carried out as described in [8]. For HPLC determination and incubation experiments with rat serum ultrafiltrate and rat serum, see [9].

Animals Female inbred NMRI mice from the Ivanovas Breeding Institute, Kisslegg, Germany; female CD2F1 mice from Zentralinstitut für Versuchstierkunde, Hannover, Germany. NMRI mice and CD2F1 mice were housed under the following conditions: room temperature 18–22 ◦C, dark and light periods of 12 h, free access to laboratory chow (Altromin 1324; Altromin GmbH, Lage, Germany) and water. Animals were randomly divided into experimental groups. Male nu/nu mice were provided by Prof. Fortmeyer University Hospital Frankfurt am Main. For details on breeding and husbandry, see: Fortmeyer H.P. Schriftenreihe Versuchstierkunde Nr.8; Paul Patrey Verlag Berlin, Hamburg 1981 [10]. Antitumor activity was assayed in female CD2F1 mice. 106 P388 mice leukemia cells were transplanted subcutaneously under the skin of the ﬂank. Therapy was started one day after tumor transplantation or 7 days after tumertransplantation, when tumor area was approximately 0.5 cm2. For the determination of tumor size, see [8], for the procedure of toxicity tests, see [4], and, for the detrmination of leukocytes, see [11].

3. Metabolism of Cyclophosphamide After the administration of CP in humans and animals, the main metabolic pathways of CP are as follows (Figure1): Sci. Pharm. 2020, 88, x FOR PEER REVIEW 3 of 12

3. Metabolism of Cyclophosphamide After the administration of CP in humans and animals, the main metabolic pathways of CP are as follows (Figure 1): After the hydroxylation of CP (1 Figure 1) by hepatic P450 enzymes, the resulting OHCP (2 Figure 1) forms an equilibrium with his tautomeric aldehyde ALDO (3 Figure 1). OHCP and ALDO are not cytotoxic. In vivo the tautomers OHCP and ALDO are irreversibly inactivated by liver enzymes. OHCP is converted by enzymatic oxidation into the non-cytotoxic KCP (7 Figure 1), and ALDO is detoxified by aldehyde dehydrogenases into the cytotoxically inactive CARB (8 Figure 1) In cell culture experiments the part of ALDO that has not been detoxified to CARB is decomposed by β elimination of acrolein (9 Figure 1) to PAM (4 Figure 1) This reaction is catalyzed by secondary phosphate ions and to a lesser extent by bicarbonate ions [12]. In vivo, however, ALDO is enzymatically decomposed by phosphodiesterases (PDE) to HPA (5 Figure 1) and PAM [9]. PAM is the alkylating moiety which is thought to unfold cell toxicity by DNA alkylation. A small part of Sci.CP Pharm. is converted2020, 88, 42 to dechlorocyclophosphamide (6 Figure 1) by side-chain hydroxylation. Toxic3 of 13 chloroacetaldehyde (10 Figure 1) is produced as a by-product.

FigureFigure 1. 1.Main Main metabolic metabolic pathways pathways ofof cyclophosphamidecyclophosphamide (for (for details details,, see see text text).). 1 1cyclophosphamidecyclophosphamide (CP),(CP),2 4-hydroxycyclophosphamide 2 4-hydroxycyclophosphamide (OHCP), 3 aldophosphamide(OHCP), 3 aldophospham (ALDO), 4 phosphoreamidemustardide (ALDO), 4 (PAM),phosphoreamidemustard5 hydroxypropanal (HPA),(PAM),6 5dechlorocyclophosphamide, hydroxypropanal (HPA), 6 7dechlorocyclophosphamide,4-ketocyclophosphamide (KCP), 7 4- 8 ketocyclophosphamide (KCP), 8 carboxyphosphamide (CARB), 9 acrolein, 10 chloroacetaldehyde. carboxyphosphamide (CARB), 9 acrolein, 10 chloroacetaldehyde.

4. PartAfter 1: the Causes hydroxylation of Toxicity of and CP (Mechanism1 Figure1) by of hepatic Action P450of Cycloph enzymes,osphamide the resulting OHCP ( 2 Figure1) forms an equilibrium with his tautomeric aldehyde ALDO (3 Figure1). OHCP and ALDO are not 4.1. Balance of the Cyclophosphamide Metabolism cytotoxic. In vivo the tautomers OHCP and ALDO are irreversibly inactivated by liver enzymes. OHCPFigure is converted 2 shows by the enzymatic blood level oxidation curves of intoCP, OHCP the non-cytotoxic and of the detoxification KCP (7 Figure products1), and KCP ALDO and is detoxiﬁedCARB after by aldehydethe intravenous dehydrogenases injection of into 100 the mg/ cytotoxicallykg CP in female inactive NMRI CARB mice (8. FigureThe detoxification1) productsIn cell cultureKCP and experiments CARB accumulate the part because of ALDO they that are has eliminated not been more detoxiﬁed slowly to from CARB the is blood decomposed than byOHCP.β elimination of acrolein (9 Figure1) to PAM ( 4 Figure1) This reaction is catalyzed by secondary phosphateHowever, ions and more to aimportant lesser extent than by these bicarbonate pharmacokinetic ions [12]. parametersIn vivo, however, is the question ALDO ishow enzymatically much of the CP administered is hydroxylated in the liver by the P450 enzyme system to OHCP and how much decomposed by phosphodiesterases (PDE) to HPA (5 Figure1) and PAM [ 9]. PAM is the alkylating of the OHCP formed escapes detoxification to KCP and CARB and is used for therapeutic and toxic moiety which is thought to unfold cell toxicity by DNA alkylation. A small part of CP is converted reactions. This question was answered in separate experiments [8]. The results are summarized in to dechlorocyclophosphamide (6 Figure1) by side-chain hydroxylation. Toxic chloroacetaldehyde Table 1. (10 Figure1) is produced as a by-product.

4. Part 1: Causes of Toxicity and Mechanism of Action of Cyclophosphamide

4.1. Balance of the Cyclophosphamide Metabolism Figure2 shows the blood level curves of CP, OHCP and of the detoxiﬁcation products KCP and CARB after the intravenous injection of 100 mg/kg CP in female NMRI mice. The detoxiﬁcation products KCP and CARB accumulate because they are eliminated more slowly from the blood than OHCP. Sci. Pharm. 2020, 88, 42 4 of 13 Sci. Pharm. 2020, 88, x FOR PEER REVIEW 4 of 12

FiFiguregure 2. BloodBlood level level curves curves of ofCP CP (○), ( OHCP), OHCP (●) (and) and detoxification detoxiﬁcation products products KCP KCP plus plus CARB CARB (▲) • (afterN) after intravenous intravenous injection injection of 100 of mg 100/kg mg# CP/kg in CP female in female NMRI NMRI mice. mice.CP is CPeliminated is eliminated from the from blood the withblood a withhalf- alife half-life of 6 min. of 6 This min. corresponds This corresponds to the tohalf the-life half-life of the offormation the formation of OHCP of OHCP of 5 min of 5. min.The Thedetoxification detoxiﬁcation products products KCP KCPand CARB and CARB accumulate accumulate because because they theyare eliminated are eliminated from fromthe blood the blood with withelimination elimination half-lives half-lives of 41 of and 41 and20 min 20 minmore more slowly slowly than than OHCP OHCP for forwhich which an elimination an elimination half half-life-life of 16of 16min min was was calculated. calculated.

TabHowever,le 1. Balance more importantof cyclophophamide than these metabolism pharmacokinetic in female parameters NMRI mice is the after question the intravenous how much of the CPadministration administered of is100 hydroxylated mg/kg cyclophosphamide in the liver bycalculated the P450 from enzyme the mean system of 5– to8 female OHCP NMRI and how mice. much of theThe OHCP 2nd column formed in escapes the % column detoxiﬁcation indicates tohow KCP much and of CARBthe OHCP and formed is used is for oxidized therapeutic to KCP and and toxic reactions.Carb. This question was answered in separate experiments [8]. The results are summarized in Table1. Bioavailability Table 1. Balance of cyclophophamideCompound metabolism nmol/g in female NMRI% mice after the intravenous administration of 100 mg/kgcyclophosphamide cyclophosphamide calculated358 from 100 the mean of 5–8 female NMRI mice. The 2nd column in the % columnOHCP indicates + ALDO how much of the328 OHCP formed92 100 is oxidized to KCP and Carb. KCP + CARB 266 74 80 Bioavailability protein binding Compound nmol/g62 17 20 % therap., toxic. reactions cyclophosphamide 358 100 Table 1 showsOHCP that+, ALDOof 358 nmol/g corresponding 328 to 92100 mg/kg CP 100 after intravenous KCP + CARB 266 74 80 administration in female NMRI mice, 92% are converted into OHCP. In total, 80% of the formed protein binding OHCP are detoxified to KCP and CARB, so that62, ultimately, 20% 17 of the CP converted 20 into OHCP is therap., toxic. reactions available for protein binding, therapeutic and toxic reactions. If renal excretion is neglected, a maximum of 8% of the applied CP is converted into chloroacetaldehyde (10, Figure 1) and dechlorocyclophosphamideTable1 shows that, of 358 (6 nmol, Figure/g corresponding 1) by side chain to 100hydroxylation. mg/kg CP after intravenous administration in femaleThis result NMRI is mice, important 92% are for converted the determination into OHCP. of t Inhe total, metabolite 80% of responsible the formed for OHCP the toxicity are detoxiﬁed of CP. CP,to KCP KCP and and CARB, CARB soare that, nontoxic ultimately, inert compounds, 20% of the CPand converted acrolein, which into OHCP is often is discussed available as for the protein main causebinding, of therapeuticthe toxicity andof CP, toxic can reactions. be ruled If out renal because excretion it is is not neglected, produced a maximum in vivo [ of9]. 8% Therefore, of the applied only OHCP,CP is converted ALDO, HPA, into chloroacetaldehyde PAM and chloroacetaldehyde (10, Figure1 are) and candidates dechlorocyclophosphamide for CP toxicity (see (Figure6, Figure 1). 1HPA) by isside also chain quite hydroxylation. out of the question as a cause of CP toxicity. It has proven to be non-toxic to eukaryotes and isThis used result as food is important additive forto prevent the determination spoilage by of growth the metabolite of pathogens responsible because for it the is active toxicity against of CP. bacterial,CP, KCP andviruses CARB and are fungi. nontoxic Neurotoxic inert compounds, and nephrotoxic and acrolein, chloroacetaldehyde which is often discussedcannot be asavoided the main as longcause as of the the P450 toxicity enzyme of CP, system can be ruled is involved out because in the it metabolism is not produced of OX.in vivo Candidates[9]. Therefore, for the only toxicity OHCP, of ALDO,CP are therefore HPA, PAM OHCP and, chloroacetaldehyde ALDO and PAM are candidates for CP toxicity (see Figure1). HPA is also quite out of the question as a cause of CP toxicity. It has proven to be non-toxic to eukaryotes and is 4.2.used The as foodReason additive of CP Toxicity to prevent spoilage by growth of pathogens because it is active against bacterial,

Sci. Pharm. 2020, 88, 42 5 of 13 viruses and fungi. Neurotoxic and nephrotoxic chloroacetaldehyde cannot be avoided as long as the P450 enzyme system is involved in the metabolism of OX. Candidates for the toxicity of CP are therefore OHCP, ALDO and PAM

4.2. The Reason of CP Toxicity Table2 shows the results of acute toxicity experiments with OHCP and PAM in male nude mice. The experiment shows that OHCP, the LD50 value of which was calculated from these data to be 530 µmol/kg (143 mg/kg), is more toxic than PAM, the LD50 of which was calculated to be 810 µmol/kg (183 mg/kg). Because substances injected intraperitoneally must pass the liver before entering the systemic circulation only a part of a drug that is metabolized in the liver enters the systemic circulation (first pass effect). Since OHCP/ALDO is metabolized in the liver to the non-toxic KCP and CARB, the difference in toxicity between OHCP and PAM is greater than that shown by the toxicity experiment.

Table 2. Acute toxicity of OHCP and PAM in male nude mice, single intraperitoneal injection, observation period 30 days.

LD50 (µmol/kg) OHCP 437 480 531 708 ~530 (µmol/kg) mortality 0/7 1/8 5/10 10/10 PAM (µmol/kg) 548 685 805 973 ~810 mortality 0/6 0/6 11/21 6/6

An indication of deviation of the real toxicity of OHCP from the measured toxicity are given from the already shown studies to the balance of the CP metabolism. The studies—Table1—show that only a maximum of 20% of the OHCP formed after CP administration is available for toxic reactions. From this, it follows that the real toxicity of OHCP is about 3 to 5 times greater than the toxicity test indicates. As will be shown in Section 5.1, OHCP is responsible for the toxicity of the tautomeric forms OHCP/ALDO.

4.3. The Mechanism of Action of Cyclophosphamide

When OHCP is incubated in rat serum ultrafiltrate pH 7, 37 ◦C, a stable equilibrium between OHCP and ALDO occurs. The half-life for the decrease in concentration of the pair of tautomers OHCP/ALDO is about 23 h. If the same experiment is done with whole rat serum, then the OHCP/ALDO equilibrium also sets in, but the half-life for the decrease in concentration of OHCP/ALDO is only 20 min [9]. This faster decrease in concentration in rat serum than in the rat serum, which has been made free of proteins, indicates that the decomposition of OHCP/ALDO is enzyme catalyzed. The splitting enzyme was identified to be a phosphodiesterase (PDE) [13]. The Michaelis constant for the enzyme in human serum was determined to be 1 mM. HPLC analysis of the serum samples with OHCP after protein precipitation and treatment with 2,4-dinitrophenylhydrazine showed time-dependent increasing concentrations of the 2,4-dinitrophenylhydrazones of 3-hydroxypropanal [9]. Based on these findings, it was concluded that the decomposition reaction of ALDO in whole rat serum is not the spontaneous β-elimination of acrolein but HPA is formed according to the reaction sequence highlighted in bold in Figure1.

4.4. The Special Feature of Cyclophosphamide Compared to Other Alkylating Agents When an alkylating substance acts on a cell, only a few of the possible alkylation reactions are therapeutically effective. A measure of the therapeutic effect is the therapeutic index—that is, the ratio from the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity. According to Brock [14] and Brock and Hohorst [15], the therapeutic index—measured in Yoshida ascites sarcoma-bearing rats—was determined to be 120 for OHCP but only 3.5 for PAM and Sci. Pharm. 2020, 88, 42 6 of 13

2.5 for nitrogen mustard. This result indicates that DNA alkylations by PAM released from ALDO in vivo are much more efficient than DNA alkylations produced by bare PAM injected. Sci. Pharm.In 9L 20 cells,20, 88, overexpressing x FOR PEER REVIEW the anti-apoptotic Bcl-2 protein, with OHCP Schwartz and Waxman6 [of16 12] could only determine a cytostatic effect but no cytotoxic apoptosis. These findings demonstrate that and, secondly, the damaged cell is eradicated by p53 mediated apoptosis. These findings suggest that the mechanism of action of OHCP proceeds in two steps. First occurs DNA damage by PAM and, the second step is responsible for the more than 30 fold higher therapeutic index of OHCP. secondly, the damaged cell is eradicated by p53 mediated apoptosis. These findings suggest that the In the following, it is shown—supported by scientific literature—that HPA strongly enhances second step is responsible for the more than 30 fold higher therapeutic index of OHCP. the apoptosis of cells, the DNA of which is damaged by PAM, which is the reason for the high In the following, it is shown—supported by scientific literature—that HPA strongly enhances the therapeutic index of OHCP compared to PAM. apoptosis of cells, the DNA of which is damaged by PAM, which is the reason for the high therapeutic index of OHCP compared to PAM. 4.5. Scientific Evidence for the Enhancement of PAM-Induced Apoptosis by HPA 4.5. ScientificHPA is Evidenceproduced for theby EnhancementLactobacillus of PAM-Inducedreuteri and Apoptosissubmitted by HPAto the culture medium [17]. ExperimentsHPA is produced by Iyer [18 by], Lactobacillus who investigated reuteri the and effects submitted of the to supernatant the culture mediumof L. reuteri [17]. cultures Experiments (LR) byon Iyertumor [18 ],necrosis who investigated factor activated the eff ectsapoptose of the supernatantsignaling pathways of L. reuteri in human cultures leukemia (LR) on tumor cells, necrosisshowed factorthat reuterin activated inhibits apoptose the signalingformation pathways of the anti in- humanapoptotic leukemia proteins cells, Bcl showed-2 and Bcl that-xL reuterin and the inhibits TNF- thedependent formation NF of-κ theB activation anti-apoptotic by inhibition proteins Bcl-2of the and translocation Bcl-xL and of the the TNF-dependent p65 subunit of NF- NFκ-BκB activation into the bynucleus. inhibition The experiments of the translocation of Iyer et of al. the further p65 subunit showed of that NF- theκB degradation into the nucleus. of IκB The is suppressed experiments by of a Iyerlack etof al.ubiquitination further showed by LR. that Thus, the degradation by the inhibition of IκB of is the suppressed nuclear translocation by a lack of ubiquitination of NF-κB, apoptosis by LR. Thus,is enhanced by the inhibition[19]. In further of the experiments, nuclear translocation Iyer et al. of NF-showedκB, apoptosis that certain is enhanced branches [19of]. the In furtherMAPK experiments,signaling pathways Iyer et are al. showedmodified that by certainLR. They branches found increased of the MAPK phosphorylation signaling pathways of the p38 are modifiedand JNK byproteins, LR. They indicating found increased activation phosphorylation of apoptosis and of thecell p38cycle and arrest. JNK proteins,In addition, indicating they measured activation the of apoptosisdecreasing and phosphorylation cell cycle arrest. of ERK, In addition, which is they a sign measured of decreased the decreasing proliferation, phosphorylation cell division, and of ERK, cell whichdifferentiation. is a sign of decreased proliferation, cell division, and cell differentiation.

4.6. Reaction Scheme for the Mechanism of Action of Oxazaphosphorine Cytostatics Involving HPA The discovery of HPA as a CP metabolite [9], the scientific scientiﬁc work of Schwarz and and Waxman Waxman [ 16], who have demonstrated that the function of PAM is to induce cytotoxic apoptosis by DNA DNA alkylation alkylation,, and the results published by Iyer and coworkers [ 18], showing showing that that HPA HPA is a proapoptotic aldehyde aldehyde,, lead toto thethe schemescheme forfor thethe mechanismmechanism ofof actionaction ofof CPCP andand otherother OXOX shownshown inin FigureFigure3 3. .

Figure 3.3. Mechanism ofof actionaction of of oxazaphosphorine oxazaphosphorine cytostatics cytostatics (OX) (OX shown) shown here here as anas examplean example for CPfor (forCP (for details, details see, text).see text).

CP is hydroxylated by P450 enzymes in the liver to OHCP OHCP,, which is in equilibrium with ALDO, ALDO isis the the pharmacologic pharmacologic active active metabolite. metabolite. A part ofA ALDOpart isof oxidizedALDO byis aldehydeoxidized dehydrogenase by aldehyde todehydrogenase the non-toxic CARBto the (not non shown).-toxic CARB The amount (not ofshown). ALDO The not oxidizedamount isof decomposed ALDO not by oxidized PDE to the is decomposed by PDE to the alkylating PAM and HPA. PAM damages DNA by alkylation. The alkylated DNA is either repaired immediately or—if this is not possible—the tumor suppressor protein p53 is activated, which induces cell cycle stop to give the cell time to repair the damage. If DNA repair is not possible, p53 induces apoptosis, which is—and this is special for CP and other OX—enhanced by HPA, which supports apoptosis by inhibiting the anti-apoptotic proteins Bcl-2 and

Sci. Pharm. 2020, 88, x FOR PEER REVIEW 7 of 12

Bcl-xL, and by inhibiting NFκB activation. Additional, HPA promotes apoptosis by enhancing mitogen activated protein kinase (MAPK) activity by enhancing JNK and p38 phosphorylation.

4.7. Conclusions from the Studies on the Toxicity and the Mechanism of Action of Cyclophosphamide for the Development of a New Generation of Oxazaphosphorine Cytostatics The consequences of the studies on toxicity and the mechanism of action of OX for the development of a new generation of OX are a) to avoid the unnecessary toxicity in the formation of OHCP through the formation of the toxic by-product chloroacetaldehyde and avoidance of toxicity of OHCP itself and b) increase the efficiency of cytotoxic apoptosis initiated by DNA damage by Sci.PAM. Pharm. 2020, 88, 42 7 of 13

5. Part 2: Bypassing Toxicity and Increasing Cytotoxic Apoptosis of Oxazaphosphorine Cytostatics alkylating PAM and HPA. PAM damages DNA by alkylation. The alkylated DNA is either repaired immediately5.1. Decrease in or—if Toxicity this isby notBypassing possible—the the Formation tumor of suppressor 4-Hydroxycyclophosphamide protein p53 is activated, in the Formation which induces of cellAldophosphamide cycle stop to give the cell time to repair the damage. If DNA repair is not possible, p53 induces apoptosis, which is—and this is special for CP and other OX—enhanced by HPA, which supports apoptosisIn order by inhibiting to determine the anti-apoptotic the proportion proteins of OHCP Bcl-2 to and the Bcl-xL, total toxicity and by inhibitingof CP, toxicity NFκB tests activation. were Additional,carried out HPAwith promotes4-(S-ethanol) apoptosis-cyclophosphamide by enhancing ( mitogen11 Figure activated 4) and the protein thiazolidine kinase (MAPK)from ALDO activity (12, byn = enhancing 1 Figure 4) JNK and andthe perhydrothiazine p38 phosphorylation. from ALDO (12, n = 2 Figure 4). The results are summarized in Table 3. 4.7. Conclusions from the Studies on the Toxicity and the Mechanism of Action of Cyclophosphamide for the DevelopmentTable 3. of aAcute New Generationtoxicity of of4 Oxazaphosphorine-(S-ethanol)-cyclophosphamide Cytostatics (11 Figure 4), aldophosphamide Thethiazolidine consequences (12, n = of1 Figure the studies 4) and on aldophosphamide toxicity and the mechanismperhydrothiazine of action (12, n of = OX2 Figure for the 4) developmentin female NMRI mice, single intraperitoneal injection, observation period 30 days. of a new generation of OX are a) to avoid the unnecessary toxicity in the formation of OHCP through the formation of the toxic by-product chloroacetaldehyde andDosage avoidance (mmol/kg) of toxicity LD50 of OHCP (mmol/kg) itself and b) increase the eﬃciency of cytotoxic apoptosis initiated by DNA damage by PAM. 4-(S-ethanol)-CP (11 Figure 4) 0.53 0.64 0.78 0.94 ~0.7 5. Partmortality 2: Bypassing Toxicity and Increasing Cytotoxic Apoptosis0/5 2/5 of3/5 Oxazaphosphorine 5/5 Cytostaticsaldophosphamid thiazolidine (12, n = 1 Figure 4) 4.9 5.9 8.9 ~6 5.1. Decreasemortality in Toxicity by Bypassing the Formation of 4-Hydroxycyclophosphamide0/5 3/7 5/5 in the Formation of Aldophosphamide4aldophosphamid perhydrothiazine (12, n = 2 Figure 4) 4.5 5.5 ~5 mortalityIn order to determine the proportion of OHCP to the total0/5 toxicity5/5 of CP, toxicity tests were carried out with 4-(S-ethanol)-cyclophosphamide (11 Figure4) and the thiazolidine from ALDO ( 12, n = 1 Figure4-4(S)- andethanol) the perhydrothiazine-cyclophosphamide) from is ALDO7 to 9 times ( 12, nmore= 2 Figuretoxic than4). The aldophosphamide results are summarized-thiazolidine in Tableand aldophosphamide3. -perhydrothiazine, which hydrolyze directly to ALDO bypassing OHCP.

Figure 4. Hydrolysis of 4-(S-ethanol)-cyclophosphamide (11) via OHCP and direct hydrolysis of aldophosphamide thiazolidine (12 n = 1) and aldophosphamide perhydrothiazine (12 n = 2) to aldophosphamide (3) and enzymatic cleavage of aldophosphamide to phosphoreamidemustard (4) and hydroxypropanal (5). Sci. Pharm. 2020, 88, 42 8 of 13

Table 3. Acute toxicity of 4-(S-ethanol)-cyclophosphamide (11 Figure4), aldophosphamide thiazolidine (12, n = 1 Figure4) and aldophosphamide perhydrothiazine ( 12, n = 2 Figure4) in female NMRI mice, single intraperitoneal injection, observation period 30 days.

Dosage (mmol/kg) LD50 (mmol/kg) 4-(S-ethanol)-CP (11 Figure4) 0.53 0.64 0.78 0.94 ~0.7 mortality 0/5 2/5 3/5 5/5 aldophosphamid thiazolidine (12, n = 1 Figure4) 4.9 5.9 8.9 ~6 mortality 0/5 3/7 5/5 Sci. Pharm. 2020, 88, x FOR PEER REVIEW 8 of 12 4aldophosphamid perhydrothiazine (12, n = 2 Figure4) 4.5 5.5 ~5 mortality 0/5 5/5 Figure 4. Hydrolysis of 4-(S-ethanol)-cyclophosphamide (11) via OHCP and direct hydrolysis of aldophosphamide thiazolidine (12 n = 1) and aldophosphamide perhydrothiazine (12 n = 2) to 4-(S-ethanol)-cyclophosphamide)aldophosphamide (3) and enzymatic iscleavage 7 to 9 timesof aldophosphamide more toxic than to phosphoreamidemustard aldophosphamide-thiazolidine (4) and aldophosphamide-perhydrothiazine,and hydroxypropanal (5). which hydrolyze directly to ALDO bypassing OHCP. This experiment shows, impressively, that, by avoiding the formation of OHCP, the toxicity is drasticallyThis reduced.experiment shows, impressively, that, by avoiding the formation of OHCP, the toxicity is drasticallyThe thiazolidines reduced. and perhydrothiazines from ALDO thus meet the ﬁrst requirement for the developmentThe thiazolidines of a new generation and perhydrothiazines of OX. They are from considerably ALDO thus less meet toxic the than first the requirement parent substance for the CP. development of a new generation of OX. They are considerably less toxic than the parent substance Whether they are also candidates for the chemotherapy of tumor diseases was examined by comparative CP. Whether they are also candidates for the chemotherapy of tumor diseases was examined by therapy experiments with the clinically proven OX Ifosfamide (IF) and the perhydrothiazine from comparative therapy experiments with the clinically proven OX Ifosfamide (IF) and the I-aldophosphamide ((IAP) 15 Figure5). IAP hydrolyzes to I aldophosphamide (IALDO) 16 Figure5)) perhydrothiazine from I-aldophosphamide ((IAP) 15 Figure 5). IAP hydrolyzes to I and L-homocysteine (18 Figure5). IALDO is split by PDE into I phosphoreamide mustard ((IPAM) 17 aldophosphamide (IALDO) 16 Figure 5)) and L-homocysteine (18 Figure 5). IALDO is split by PDE Figure5) and HPA ( 5 Figure5). into I phosphoreamide mustard ((IPAM) 17 Figure 5) and HPA (5 Figure 5).

COO- R + R H3N R - CHO COO HO HS 18 5 Cl O NH HN Cl O NH NH N P N P Cl O CHO P H O S H O N 15 16 17 H OH

IAP: R = Cl SUM-IAP: R= -OSO2CH3

FigureFigure 5. 5.Hydrolysis Hydrolysis ofof I-aldophosphamideI-aldophosphamide ( (IAP)IAP) (R (R == Cl)Cl) and and SUM SUM-IAP-IAP (R (R= –=OSO–OSO2CH2CH3 (153))( 15to)) I- to I-aldophosphamidealdophosphamide ( (1616) )and and homocysteine homocysteine (18 ()18 and) and the theformation formation of I-phosporeamidemustard of I-phosporeamidemustard (17) and (17 ) and3-hydroxypropanal 3-hydroxypropanal (5).( 5).

TheThe experiment experiment was was carried carried outout withwith I-aldophosphamideI-aldophosphamide perhydrothiazine perhydrothiazine (IAP) (IAP) because because the the synthesissynthesis and and handling handling of of the the strongly strongly hygroscopichygroscopic andand amorphous aldophosphamide aldophosphamide thiazolidine thiazolidine waswas problematic. problematic. In In contrast, contrast, the the synthesis synthesis of IAPof IAP is not is not a problem. a problem. The The substance substance was was used used as a stable,as a readilystable, water-soluble readily water- lyophylizatesoluble lyophylizate for the followingfor the following experiments. experiments. TheThe results results of of therapy therapy experiments experiments in in P388 P388 tumor-bearing tumor-bearing female female CD2F1 CD2F1 mice mice are aresummarized summarized in Tablein Table4. 4.

Table 4. Antitumor activity of equitoxic dosages of Ifosfamide and IAP in P388 tumor-bearing mice. Therapy was started on day 1 after 106 P388 tumor cells were subcutaneously transplantated in female CD2F1 mice. Ifosfamide was administered intraperitoneally, IAP was administered subcutaneously. 1 increase in life span (ILS), 2 surviving time > 100 d.

Dosage (mg/kg) ILS (%) 1 Long Time Survivors 2 175 d1-4 233 0/5 Ifosfamide 350 d1-2 289 0/5 750 d1-4 244 2/6 IAP (R = Cl, 15 Figure 5) 1500 d1-2 400 4/5

Therapy with IAP cures 2/6 or 4/5 animals (surviving time > 100 d) while only survival time extensions are achieved with the same equitoxic dose IF. This experiment demonstrates the superiority of IAP that can be dosed higher than IF because it forms IALDO directly by bypassing the toxic OHIF and is therefore much less toxic than IF.

5.2. Increase in the Antitumor Activity of I-Aldoperhydrthiazines by Modulating the Alkylating Function

Sci. Pharm. 2020, 88, 42 9 of 13

Dosage (mg/kg) ILS (%) 1 Long Time Survivors 2 175 d1-4 233 0/5 Ifosfamide 350 d1-2 289 0/5 750 d1-4 244 2/6 IAP (R = Cl, 15 Figure5) 1500 d1-2 400 4/5

5.2. Increase in the Antitumor Activity of I-Aldoperhydrthiazines by Modulating the Alkylating Function According to the proposed mechanism of action of CP and other OX, DNA damage caused by PAM or IPAM is not the cause of cell death; it merely has the function of initiating cytotoxic apoptosis. Since cellular DNA repair mechanisms repair DNA damage very eﬃciently and quickly, only a small part of the DNA damage caused by the alkylating metabolites trigger apoptosis. In order to increase this proportion, it is necessary to generate DNA damage that cannot be repaired or is diﬃcult to repair. The alkylating function of all OX consists of 2 chloroethyl groups, which generate easily repairable DNA inter strand crosslinks. Therefore, most of the DNA damage caused by the currently used OX is repaired before it initiates apoptosis. According to Povirk and Shuker [20] mesyl-ethyl groups form intra strand cross-links that are poorly repaired (http://www.atdbio.com/content/16/Nucleic-acid-drug-interactions, accessed on 01 March 2018) Figure6 shows the results of therapy experiments with IAP and an IAP derivative—SUM-IAP—in which a chloroethyl group of IAP is substituted by a mesyl ethyl group (R = –OSO2CH3, 15 Figure5). A small dose of IAP and SUM-IAP of 0.29 mmol/kg was administered on day 7–11 after tumor transplantation when the tumor area was approximately 0.5 cm2. The experiment with IAP, which produces easily repairable inter strand crosslinks, shows only a marginal growth delay of the tumor, whereas, in the experiment with SUM-IAP that generates poorly repairable DNA intra strand cross links, the tumor burden is reduced below detection level for 8 days before tumor cells that have become resistant grow again. In a further experiment (not shown) with only one injection of 1.32 mmol/kg IAP and SUM-IAP (521, respectively, 600 mg/kg), the result was in principle the same, only growth delay in the experiment with IAP but decrease in tumor burden below detection level in the experiment with SUM-IAP. Sci. Pharm. 2020, 88, x FOR PEER REVIEW 9 of 12

According to the proposed mechanism of action of CP and other OX, DNA damage caused by PAM or IPAM is not the cause of cell death; it merely has the function of initiating cytotoxic apoptosis. Since cellular DNA repair mechanisms repair DNA damage very efficiently and quickly, only a small part of the DNA damage caused by the alkylating metabolites trigger apoptosis. In order to increase this proportion, it is necessary to generate DNA damage that cannot be repaired or is difficult to repair. The alkylating function of all OX consists of 2 chloroethyl groups, which generate easily repairable DNA inter strand crosslinks. Therefore, most of the DNA damage caused by the currently used OX is repaired before it initiates apoptosis. According to Povirk and Shuker [20] mesyl-ethyl groups form intra strand cross-links that are poorly repaired (http://www.atdbio.com/content/16/Nucleic -acid-drug -interactions, accessed on 01.March.2018) Figure 6 shows the results of therapy experiments with IAP and an IAP derivative—SUM-IAP— in which a chloroethyl group of IAP is substituted by a mesyl ethyl group (R = –OSO2CH3, 15 Figure 5). A small dose of IAP and SUM-IAP of 0.29 mmol/kg was administered on day 7–11 after tumor transplantation when the tumor area was approximately 0.5 cm2. The experiment with IAP, which produces easily repairable inter strand crosslinks, shows only a marginal growth delay of the tumor, whereas, in the experiment with SUM-IAP that generates poorly repairable DNA intra strand cross links, the tumor burden is reduced below detection level for 8 days before tumor cells that have become resistant grow again. In a further experiment (not shown) with only one injection of 1.32 mmol/kg IAP and SUM-IAP (521, respectively, 600 mg/kg), the result was in principle the same, only

Sci.growth Pharm. delay2020, 88 in, 42the experiment with IAP but decrease in tumor burden below detection level10 in of the 13 experiment with SUM-IAP.

FigureFigure 6.6. TumorTumor growthgrowth curvescurves ofof subcutaneouslysubcutaneously transplantedtransplanted P388P388 tumorstumors inin femalefemale CD2F1CD2F1 micemice followingfollowing therapytherapy withwith 0.290.29 mmolmmol IAPIAP (115(115 mgmg/kg)/kg) ((■)) oror SUM-IAPSUM-IAP (133(133 mgmg/kg)/kg) ((●)) i.p.i.p. onon dayday 7–117–11 • (arrows),(arrows), mean mean ± standardstandard deviationdeviation fromfrom 33 animals.animals. “Control”“Control” ((○)) showsshows thethe tumortumor growthgrowth curvecurve ofof ± subcutaneouslysubcutaneously transplantedtransplanted P388P388 micemice leukemialeukemia cellscells inin untreated untreated# female female CD2F1 CD2F1 mice. mice.

ToTo compare the the antitumor antitumor ac activitytivity of ofIAP IAP and and SUM SUM-IAP-IAP quantitatively, quantitatively, the tumor the tumor growth growth curves curvesafter therapy after therapy with IAP with and IAP SUM and-IAP SUM-IAP were evaluated were evaluated using usingthe Alexander the Alexander and Mikulski and Mikulski back- back-extrapolationextrapolation method method [21]. From [21]. the From slopes the of slopes the tumor of the growth tumor curves growth, it curves, can be itconcluded can be concluded that there thatis a 10 there4–10 is5 times a 104 –10increase5 times in increaseantitumor in activity antitumor when activity one 2 when-chloroethyl one 2-chloroethyl group in IAP group is substituted in IAP is substitutedby a mesyl byethyl a mesyl group ethyl [11]. group [11].

6.6. TheThe New New Generation Generation of of Oxazaphosphorine Oxazaphosphorine Cytostatics Cytostatics Are are Candidates Candidates for Immunologicalfor Immunological Tumor Therapy Tumor Therapy It is well known that CP in the lower dose range has—in addition to its antitumor activity in the higher dose range—immunomodulatory properties [22,23]. It has been shown for CP that it activates anti-tumor-driven CD8+ T-cell (CTL) responses [24], supports tumor-driven CD4+ T-cell (Th) differentiation [25] and stimulates dendritic cell maturation [26]. The fact that CP has an immune stimulating effect in low doses is due to the special sensitivity of T cell inhibiting regulatory T cells (Treg) to ALDO. Heylmann and collegues [27] compared the sensitivity of Treg, CTL and Th to Mafosfamide, which hydrolyses to OHCP within minutes. They could show that the high sensitivity of Treg compared to CTL and Th is due to induction of apoptosis and decrease in ability of Treg to repair damaged DNA. Thus, OX are antitumor substances that combine 2 mechanisms of action. These are the immediate cytotoxic apoptosis triggered by DNA alkylation and antitumor-driven CTL response. The latter, however, only with low doses. The question arises: What influence does the type of DNA damage—repairable or irreparable—have on the immunomodulatory abilities of OX? This question is answered by an experiment summarized in Figure7 and already published in [ 28]. Solid growing P388 tumor-bearing mice were treated with 666 mg/kg SUM-IAP on days 7 and 8 after tumor transplantation. This treatment causes a reduction in the tumor below detection limit from day 11 to day 22 (not shown). The tumor was then measurable again, animals also suffered from metastases and died on days 25–30 (Figure7). Sci. Pharm. 2020, 88, x FOR PEER REVIEW 10 of 12

It is well known that CP in the lower dose range has—in addition to its antitumor activity in the higher dose range—immunomodulatory properties [22,23]. It has been shown for CP that it activates anti-tumor-driven CD8+ T-cell (CTL) responses [24], supports tumor-driven CD4+ T-cell (Th) differentiation [25] and stimulates dendritic cell maturation [26]. The fact that CP has an immune stimulating effect in low doses is due to the special sensitivity of T cell inhibiting regulatory T cells (Treg) to ALDO. Heylmann and collegues [27] compared the sensitivity of Treg, CTL and Th to Mafosfamide, which hydrolyses to OHCP within minutes. They could show that the high sensitivity of Treg compared to CTL and Th is due to induction of apoptosis and decrease in ability of Treg to repair damaged DNA. Thus, OX are antitumor substances that combine 2 mechanisms of action. These are the immediate cytotoxic apoptosis triggered by DNA alkylation and antitumor-driven CTL response. The latter, however, only with low doses. The question arises: What influence does the type of DNA damage—repairable or irreparable— have on the immunomodulatory abilities of OX? This question is answered by an experiment summarized in Figure 7 and already published in [28]. Solid growing P388 tumor-bearing mice were treated with 666 mg/kg SUM-IAP on days 7 and 8 after tumor transplantation. This treatment causes Sci.a reduction Pharm. 2020 in, 88 the, 42 tumor below detection limit from day 11 to day 22 (not shown). The tumor11 was of 13 then measurable again, animals also suffered from metastases and died on days 25–30 (Figure 7).

Figure 7.7. Survival curves of CD2F1 mice bearingbearing subcutaneouslysubcutaneously transplanted solid growing P388 tumors following therapy therapy with with 666 666 mg mg//kgkg SUM SUM-IAP-IAP on on days days 7 7and and 8 8(upward (upward arrows, arrows, broken broken line, line, 3 3animals) animals) and and therapy therapy following following 666 666 mg mg/kg/kg SUM SUM-IAP-IAP on on days days 7 7and and 8 8 and and on on days days 14 and 15 (downward(downward arrows,arrows, solidsolid line,line, 55 animals)animals) andand thethe number of leucocytes following therapy on days 7.8 and 14.15 (mean values +/ SD). The median survival time of untreated tumor-bearing controls was and 14.15 (mean values +/− SD). The median survival time of untreated tumor-bearing controls was 11–1211–12 days.

In thethe samesame experiment,experiment, but but with with an an additional additional therapy therapy cycle cycle on on days days 14 14 and and 15, 15, no regrowthno regrowth of the of primarythe primary tumor tumor and no and formation no formation of metastasis of metastasis were observed. were observed. All animals All survived animals the survived observation the periodobservation of 100 period days andof 100 can days be regarded and can asbe cured,regarded concurrent as cured, a sharpconcurrent increase a sharp in no increase of leukocytes in no of is measuredleukocytes(Figure is measured7). The ( latterFigure is 7). proof The that latter the regrowthis proof that of the the primary regrowth tumor, of the regrowth primary of tumortumor, cellsregrowth that haveof tumor become cells resistant, that have and become the formation resistant of, and metastases the formation are prevented of metastases by anti-tumor-driven are prevented immunologicalby anti-tumor-driven mechanisms. immunological mechanisms. This experiment shows,shows, impressively impressively,, that by creating irreparableirreparable DNA alkylations,alkylations, the second mechanism of action of OX, the anti-tumor-drivenanti-tumor-driven stimulation of the immune system, is greatly increased. With With the new generation of OX, which were presentedpresented here, for the firstfirst time anti-tumoranti-tumor drugs are available, which can simultaneously eradicate tumor cells by cytotoxic apoptosis and by anti-tumor-drivenanti-tumor-driven immunological mechanismsmechanisms ofof action.action. Funding: This study was funded by the Bundesministerium für Forschung und Technologie. Funding: This study was funded by the Bundesministerium für Forschung und Technologie. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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