Activation of oxazaphosphorines by cytochrome P450: application to gene-directed enzyme prodrug therapy for cancer.

Cancer chemotherapeutic prodrugs, such as the oxazaphosphorines cyclophosphamide and ifosfamide, are metabolized by liver cytochrome P450 enzymes to yield therapeutically active, cytotoxic metabolites. The effective use of these prodrugs is limited by host toxicity associated with the systemic distribution of cytotoxic metabolites formed in the liver. This problem can, in part, be circumvented by implementation of cytochrome P450 gene-directed enzyme prodrug therapy (P450 GDEPT), a prodrug activation strategy for cancer treatment that augments tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating cytochrome P450 enzyme. P450 GDEPT has been exemplified in preclinical rodent and human tumor models, where chemosensitivity to a P450 prodrug can be greatly increased by introduction of a prodrug-activating P450 gene. Further enhancement of the efficacy of P450-based gene therapy can be achieved: by co-expression of P450 with the flavoenzyme NADPH-P450 reductase, which provides electrons required for P450 metabolic activity; by metronomic (anti-angiogenic) scheduling of the prodrug; by localized delivery of the prodrug to the tumor; and by combination with anti-apoptotic factors, which slow the death of the P450 'factory' cells and thereby enhance the bystander cytotoxic response. P450 GDEPT has several important features that make it a clinically attractive strategy for cancer treatment. These include: the substantial bystander cytotoxicity of P450 prodrugs such as cyclophosphamide and ifosfamide; the ability to use human P450 genes and thereby avoid an immune response to the therapeutic gene; the use of well-established conventional chemotherapeutic prodrugs, as well as bioreductive drugs activated by P450/P450 reductase in a hypoxic tumor environment; and the potential to decrease systemic exposure to active drug metabolites by selective inhibition of hepatic P450 activity. Recent advances in this area of research are reviewed, and two proof-of-concept clinical trials that highlight the utility of this strategy are discussed.     

Cytochrome P450-based cancer gene therapy: recent advances and future prospects.

Cytochrome P450-based cancer gene therapy is a novel prodrug activation strategy for cancer treatment that has substantial potential for improving the safety and efficacy of cancer chemotherapeutics. The primary goal of this strategy is to selectively increase tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating P450 enzyme. This strategy has been exemplified for the alkylating agents cyclophosphamide and ifosfamide, which are bioactivated by select P450 enzymes whose expression is generally high in liver and deficient in tumor cells. Transduction of tumors with a prodrug-activating P450 gene, followed by prodrug treatment, greatly increases intratumoral formation of activated drug metabolites. This leads to more efficient killing of the transduced tumor cells without a significant increase in host toxicity. P450 gene therapy is accompanied by substantial bystander cytotoxicity which greatly enhances the therapeutic effect by extending it to nearby tumor cells not transduced with the therapeutic P450 gene. Although endogenous P450 reductase is not expected to be a limiting factor in prodrug activation in tumor cells that express moderate levels of an exogenous P450 gene, P450 reductase transduction has recently been found to substantially enhance intratumoral prodrug activation and its associated therapeutic effects. Using this gene combination, an overall 50- to 100-fold increase in tumor cell kill in vivo over that provided by hepatic drug activation alone has been observed. Striking improvements in therapeutic effects can thus be achieved using an established anticancer drug in an intratumoral prodrug activation strategy based on the combination of a cytochrome P450 gene with the gene encoding NADPH-P450 reductase. This strategy is readily extendable to several other widely used P450-activated cancer chemotherapeutic prodrugs, as well as to prodrugs that undergo P450 reductase-dependent bioreductive activation and which may exhibit synergy when combined with P450-activated prodrugs in a P450/P450 reductase-based cancer gene therapeutic regimen.     

Cytochrome P450-based gene therapy for cancer treatment: from concept to the clinic

In the last 16 years, more than a dozen gene-directed enzyme prodrug therapies for cancer treatment have been evaluated in preclinical studies. However, only few of them have evolved to the stage of clinical trial. This review assesses current knowledge in the area of cancer gene therapy, emphasizing cytochrome p450 (CYP)-based prodrug activation systems. This approach is intuitively highly suitable for the treatment of cancers, since several major anticancer drugs are activated by liver CYP enzymes. Important features of this strategy include: 1) use of human CYP genes to avoid immune complications that may hamper expression of therapeutic genes of non-human origin and thereby inhibit prodrug activation, 2). use of well established and clinically effective anticancer prodrugs, 3). strong bystander cytotoxic effect seen with all liver-activated CYP prodrugs, 4). the potential to inhibit liver CYP activity and expression to increase the bioavailability of prodrugs for CYP-transduced tumors, 5). possible extension to many CYP enzymes and their potential anticancer prodrug substrates, and 6). it can be used to arm therapeutic conditionally replicating viruses. Historically, this strategy utilized CYP 2B1 to activate oxazaphosphorines. It is now becoming clear that the repertoire of prodrugs is expandable and that CYP gene candidates are not limited to naturally occurring CYP genes, but may also encompass engineered CYP enzymes, improved by site directed mutagenesis or other approaches. Encouraging results from a recent phase I/II clinical trial that have implemented this strategy, as well as emerging problems related to gene delivery are discussed in this review.     

Beta-glucuronidase-mediated drug release

The selective activation of a relatively non-toxic prodrug by an enzyme present only in the tumour should enhance the drug concentration at the tumour site and result in a better anti-tumour effect and a reduction in systemic toxicity as compared to conventional chemotherapy. beta-Glucuronidase is such an enzyme. It is normally expressed in the lysosomes of cells. In larger tumours, however, high levels of the enzyme are present in necrotic areas. Several glucuronide prodrugs have been synthesised that can be activated by beta-glucuronidase. They are relatively non-toxic due to their hydrophilic nature, which prevents them from entering cells and thus from contact with lysosomal beta-glucuronidase. The main problem of glucuronide prodrugs for clinical use is their fast renal clearance. Special attention should be paid to the development of new less hydrophilic prodrugs with slower clearance, as this would result in a prolonged exposure to beta-glucuronidase at the site of the tumour and a reduction of the amount of prodrug needed. A number of interesting anthracyclin-based glucuronide prodrugs have been synthesised and have shown favourable therapeutic effects compared to treatment with the parent drug. The tumoural levels of beta-glucuronidase can even be enhanced by two-step approaches, in which exogenous enzyme is targeted to the tumour by an antibody (ADEPT) or by the gene encoding the enzyme in transduced tumour cells (GDEPT). The ADEPT and GDEPT approaches in combination with glucuronide prodrugs have shown enhanced efficacy in experimental tumour models. Further improvement of ADEPT and GDEPT is warranted to optimise the tumour uptake and retention of antibody-enzyme fusion proteins and the efficiency and safety of current gene delivery methods. In conclusion, it is clear that glucuronide prodrugs hold promise for future use in the treatment of cancer in patients as monotherapy. Enhancement of the therapeutic effects of glucuronide prodrugs, also in patients with small tumour lesions, may possibly be achieved by techniques that target beta-glucuronidase specifically to the site of the tumour.     

Gene-Directed Enzyme Prodrug Therapy

As one targeting strategy of prodrug delivery, gene-directed enzyme prodrug therapy (GDEPT) promises to realize the targeting through its three key features in cancer therapy—cell-specific gene delivery and expression, controlled conversion of prodrugs to drugs in target cells, and expanded toxicity to the target cells’ neighbors through bystander effects. After over 20 years of development, multiple GDEPT systems have advanced into clinical trials. However, no GDEPT product is currently marketed as a drug, suggesting that there are still barriers to overcome before GDEPT becomes a standard therapy. In this review, we first provide a general introduction of this prodrug targeting strategy. Then, we utilize the four most thoroughly studied systems to illustrate components, mechanisms, preclinical and clinical results, and further development directions of GDEPT. These four systems are herpes simplex virus thymidine kinase/ganciclovir, cytosine deaminase/5-fluorocytosine, cytochrome P450/oxazaphosphorines, and nitroreductase/CB1954 system. Later, we focus our discussion on bystander effects including local and distant bystander effects. Lastly, we discuss carriers that are used to deliver genes for GDEPT including virus carriers and non-virus carriers. Among these carriers, the stem cell-based gene delivery system represents one of the newest carriers under development, and may brought about a breakthrough to the gene delivery issue of GDEPT.KEY WORDS: bystander effects, gene delivery, gene-directed enzyme, prodrug, stem cell-based targeting     

The Design of Selectively-activated Anti-cancer Prodrugs for use in Antibody-directed and Gene-directed Enzyme-Prodrug Therapies*

Systemic anti-proliferative agents (cytotoxins) have been the most successful single design concept for anti-cancer drugs. However, they have inherent limitations (they target dividing cells rather than cancer cells) which limit their clinical efficacy, especially toward the more slowly-growing solid tumours. New concepts are required to improve the selectivity of their killing of tumour cells. One possibility is the use of prodrugs which can be activated selectively in tumour tissue. Several potential mechanisms for this are being explored, including tumour hypoxia, low extracellular pH, therapeutic radiation and tumour-specific endogenous or exogenous enzymes. In the last approach the exogenous enzyme can be delivered by attachment to monoclonal antibodies (ADEPT) or as DNA constructs containing the corresponding gene (GDEPT). A limitation of both approaches is that only a small proportion of the tumour cells become activation-competent, but this can be substantially overcome by the design of appropriate prodrugs capable of killing activation-incompetent cells via a bystander effect. We have proposed a modular approach to prodrug design in which a trigger unit determines tumour selectivity and an effector unit achieves the desired level of killing of cells when the trigger is activated. For ADEPT and GDEPT prodrugs the primary requirement of the trigger is efficient and selective activation by the appropriate enzyme; the released effector must be a potent, diffusible cytotoxin which fully exploits the small proportion of cells capable of activating the prodrug. A wide variety of chemistries has been used, but many of the existing effectors do not have all of these properties. We report work on two types of cytotoxin derived from very potent anti-tumour antibiotics (enediynes and amino-seco-cyclopropylindolines) as effectors in prodrugs for ADEPT and GDEPT applications.     

Enzyme-catalyzed activation of anticancer prodrugs

The rationale fo the development of prodrugs relies upon delivery of higher concentrations of a drug to target cells compared to administration of the drug itself. In the last decades, numerous prodrugs that are enzymatically activated into anti-cancer agents have been developed. This review describes the most important enzymes involved in prodrug activation notably with respect to tissue distribution, up-regulation in tumor cells and turnover rates. The following endogenous enzymes are discussed: aldehyde oxidase, amino acid oxidase, cytochrome P450 reductase, DT-diaphorase, cytochrome P450, tyrosinase, thymidylate synthase, thymidine phosphorylase, glutathione S-transferase, deoxycytidine kinase, carboxylesterase, alkaline phosphatase, beta-glucuronidase and cysteine conjugate beta-lyase. In relation to each of these enzymes, several prodrugs are discussed regarding organ- or tumor-selective activation of clinically relevant prodrugs of 5-fluorouracil, axazaphosphorines (cyclophosphamide, ifosfamide, and trofosfamide), paclitaxel, etoposide, anthracyclines (doxorubicin, daunorubicin, epirubicin), mercaptopurine, thioguanine, cisplatin, melphalan, and other important prodrugs such as menadione, mitomycin C, tirapazamine, 5-(aziridin-1-yl)-2,4-dinitrobenzamide, ganciclovir, irinotecan, dacarbazine, and amifostine. In addition to endogenous enzymes, a number of nonendogenous enzymes, used in antibody-, gene-, and virus-directed enzyme prodrug therapies, are described. It is concluded that the development of prodrugs has been relatively successful; however, all prodrugs lack a complete selectivity. Therefore, more work is needed to explore the differences between tumor and nontumor cells and to develop optimal substrates in terms of substrate affinity and enzyme turnover rates fo prodrug-activating enzymes resulting in more rapid and selective cleavage of the prodrug inside the tumor cells.     

Cancer chemotherapy and drug metabolism.

Drug-metabolizing enzymes and drug transporters are key determinants of the pharmacokinetics and pharmacodynamics of many antineoplastic agents. Metabolism and transport influence the cytotoxic effects of antineoplastic agents in target tumor cells and normal host tissues. This article summarizes several state-of-the-art approaches to enhancing the effectiveness and safety of cancer therapy based on recent developments in our understanding of antineoplastic drug metabolism and transport. Advances in four interrelated research areas presented at a recent symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics (Experimental Biology 2004; Washington D.C., April 17-21, 2004) are discussed: 1) interactions of anthracyclines with drug-metabolizing enzymes; 2) use of hypoxia-selective gene-directed enzyme prodrug therapy (GDEPT) in combination with bioreductive prodrugs; 3) synergy between glutathione conjugation and conjugate efflux in conferring resistance to electrophilic toxins; and 4) use of cytochromes P450 as prodrug-activating enzymes in GDEPT strategies. A clear theme emerged from this symposium: drug metabolism and transport processes can be modulated and exploited in ways that may offer distinct therapeutic advantages in the management of patients with cancer.     

Prodrugs for antibody- and gene-directed enzyme prodrug therapies (ADEPT and GDEPT)

Antibody- and gene-directed enzyme prodrug therapy are two-step targeting strategies designed to improve the selectivity of antitumour agents. The approaches are based on the activation of specially designed prodrugs by antibody-enzyme conjugates targeted to tumour-associated antigens (ADEPT) or by enzymes expressed by exogenous genes in tumour cells (GDEPT). Herein the design, synthesis, physico-chemical and biological properties, kinetics and clinical trials of the prodrugs and the enzymes carboxypeptidase G2 and nitroreductase are reviewed for ADEPT and GDEPT.     

Gene directed enzyme prodrug therapy for ovarian cancer: could GDEPT become a promising treatment against ovarian cancer?

Gene-directed enzyme prodrug therapy (GDEPT) involves the treatment concept of having maximal efficacy and minimal adverse effects. Several GDEPT strategies have been developed combining cytosine deaminase and 5-fluorocytosine, cytochrome P450 2B1 and cyclophosphamide, and carboxylesterase (CES) and irinotecan in experimental models. The active forms of these prodrugs, however, are not a frontline therapy for the treatment of ovarian cancer. It would be beneficial to develop a more effective prodrug-enzyme combination for the treatment of this disease. Paclitaxel (Taxol; TAX) is currently one of the most important anti-cancer drugs in chemotherapy of ovarian cancer. One of TAX prodrugs, 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et), was generated and examined regarding its pharmacological aspects. The prodrug of TAX-2'-Et converts into active form TAX by carboxylesterase (CES). TAX-2'-Et did not exhibit polarized transport in the Caco-2 cells expressing P-glycoprotein (P-gp) in the absence or presence of verapamil which is a inhibitor of P-gp, suggesting that TAX-2'-Et is not a target of P-gp like TAX and rhodamine123. Moreover, SKOV3/TAX60 cells which are overexpressing P-gp did not also exhibit any change in cellular uptake of TAX-2'-Et regardless of the absence or presence of verapamil. Consequently, the uptake of TAX-2'-Et into the TAX-resistant cells was quantitatively similar to that internalized in the parental SKOV3 cells which are P-gp-negative. In the CES-transfected SKOV3 cells, the EC50 value of TAX (10.6 nM) was approximately 4-fold higher than that of TAX-2'-Et (2.5 nM). We herein provide evidence that TAX-2'-Et could circumvent P-gp-associated cellular efflux of TAX, suggesting that this combination therapy is a potential GDEPT strategy for ovarian cancer in the future. Finally, this review focuses on the development, application and potential of various GDEPTs for treating ovarian cancer, and the scope and progress of new GDEPTs are discussed.     

From bench to bedside for gene-directed enzyme prodrug therapy of cancer

Gene therapy of cancer offers the possibility of a targeted treatment that destroys tumors and metastases, but not normal tissues. In gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, the gene encoding an enzyme is delivered to tumor cells, followed by administration of a prodrug, which is converted locally to a cytotoxin by the enzyme. The producer cells as well as surrounding bystanders are subsequently killed. Promising results have meant that suicide gene therapy has reached multicenter phase III clinical trials. This review will discuss the development, efficiency, mode of action and pharmacokinetics of seven GDEPT systems in vitro and in vivo. We will review the latest data of those systems in clinical trials (herpes simplex virus thymidine kinase/gancyclovir, bacterial cytosine deaminase/5-fluorocytosine, bacterial nitroreductase/CB1954 and cytochrome P450/cyclophosphamide), as well as the development of more recent and experimental systems which are not yet in clinical trials (P450 reductase/tirapazamine, carboxypeptidase/CMDA, horseradish peroxidase/indole-3-acetic acid or paracetamol and others).     

Prodrugs in genetic chemoradiotherapy

Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility. Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes. Gene-Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect). This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed. In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization. Some, but not all GDEPT approaches have been analysed in combination with radiotherapy. Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N. Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E. coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart. Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.     

Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy

Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.     

Prodrugs: effective solutions for solubility, permeability and targeting challenges

This 2-day inaugural conference on prodrugs was presented by Pharmaceutical Education Associates and covered recent developments in prodrug techniques to solve delivery and targeting issues in drug discovery and development. The speakers were drawn from industry and academia, and the conference was attended mostly by researchers working in the pharmaceutical and biotechnology industries. A number of topics were presented at the conference, from basic prodrug design and functional group considerations to drug metabolism involving cytochrome P450 enzymes, from increasing water solubility, bioavailability, permeability and stability to tumor targeting, from the development of new anti-inflammatory agents to anti-HIV agents, and from the use of transporters and receptor-mediated endocytosis in prodrug delivery to the use of gene therapy for enzyme delivery to cancer cells and tissues. Several case studies were presented including improved pharmaceutical products in the clinic and at various stages of development.     

HepDirect prodrugs for targeting nucleotide-based antiviral drugs to the liver

HepDirect prodrugs represent a novel class of cytochrome P450-activated prodrugs capable of targeting certain drugs to the liver. In this review, the HepDirect prodrug concept and its use for the delivery of nucleotides to the liver for the treatment of viral hepatitis is summarized. Preclinical and clinical data for the most advanced HepDirect prodrug, pradefovir, highlight the liver-targeting capability of these prodrugs, and the potential benefit of liver targeting on drug efficacy, safety and viral resistance.     

Targeted prodrug design to optimize drug delivery

Classical prodrug design often represents a nonspecific chemical approach to mask undesirable drug properties such as limited bioavailability, lack of site specificity, and chemical instability. On the other hand, targeted prodrug design represents a new strategy for directed and efficient drug delivery. Particularly, targeting the prodrugs to a specific enzyme or a specific membrane transporter, or both, has potential as a selective drug delivery system in cancer chemotherapy or as an efficient oral drug delivery system. Site-selective targeting with prodrugs can be further enhanced by the simultaneous use of gene delivery to express the requisite enzymes or transporters. This review highlights evolving strategies in targeted prodrug design, including antibody-directed enzyme prodrug therapy, genedirected enzyme prodrug therapy, and peptide transporter-associated prodrug therapy.     

Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k_cat/K_m) and when over-expressed in an E. colinfsA⁻/nfsB⁻ gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.     

Novel anthracycline prodrugs

This paper highlights recent patents in the field of anthracycline prodrugs, which are employed in tumour-selective chemotherapy. The prodrugs can be a part of a two-step directed enzyme prodrug therapy (DEPT), which involves the localisation of the prodrug trigger at the tumour site, followed by the administration of the prodrug and subsequent tumour-selective anthracycline release. In most cases this trigger is an enzyme, which is indirectly localised by an antibody (ADEPT) or a gene encoding for an enzyme (GDEPT). Furthermore, anthracyclines can be targeted to the tumour site via prodrug monotherapy. Anthracycline prodrugs exploiting differences in physiological conditions, such as a lower pH and a lower oxygen tension in tumour tissue compared to healthy tissue, tumour-specific enzymes, such as plasmin, cathepsin B and β-glucuronidase are discussed. Finally, prodrugs are reviewed that home to tumour-selective receptors. Promising advances in this field concern receptors that are required for angiogenesis.