Methotrexate – Pci 32765 Chemical

The pharmacology of methotrexate

Kurt Barnhart, Christos Coutifaris & Melissa Esposito

Dctopic pregnancy (EP) is a major cause of maternal morbidity and mortality. The treatment of this condition is primarily surgical, but medical management in selected cases is safe, effective, cost-effective and eliminates the morbidity of surgery. Methotrexate (MTX) is afolate antago- nist that can be used fornon-oncologic purposes including the treatment of EP. The dose and duration of MTX therapy for EP is much lower than that used in oncology cases, thus reducing side effects and increasing safety. MTX selectively acts on rapidly dividing cells, such as trophoblast cells which comprise the implantation site of the early gestation. The two most common methods of administering MTXto patients with EP are im. admini- stration of a single-dose, based on body surface area and calculated by the equation 50 mg/m (without the need for leucovorin rescue), or the multiple-dose regimen of 1 mg/kg of MTX, alternating with 0.1 mg/kg of leucovorin rescue. Both methods have asimilar side effect profile, resulting in the rare occurrence of nausea, vomiting, stomatitis, elevated liver function tests, anorexia and diarrhoea. The two methods yield success rates similar to those of conservative surgical therapy with similar future fertility. The potential single- and multi-dose methods have never been directly compared, but it appears that the success of multiple dosing is more effective. As the efficacy of MTX therapy is not 100%, women must be followed clinically until there is compete resolution of human Chorionic Gonadotropin (hCG) titers from their serum.
Keywords: ectopic pregnancy, medical management, methotrexate, non-oncologic use of chemotherapy

Exp. Opin. Pharmacother. (2001) 2(3):409-417

1. Introduction
Methotrexate (MTX) is afolate antagonistthatin recentyears has been used increasingly fornon-oncologic purposes, including the treatmentof ectopic pregnancy (EP). EP is defined as implantation of an embryo outside the uterus, most commonly in the fallopian tube. An EP can result in rupture of the fallopian tube and intra-abdominal haemorrhage. The number of EPs diagnosed in the United States has increased from17,800in 1970to 108,000 in 1992; a 6-fold increase in the past two decades [1]. EP now accounts for 2% of all pregnancies in the United States [1]. EP is the leading pregnancy- related cause of death in the firsttrimesterof pregnancy [1]and is, therefore, a major public health concern. Currently, it accounts for 9 – 13% of all pregnancy deaths [2,3]. Even if EP is diagnosed and treated before rupture of the fallopian tube, its damage results in agreater risk of future EP which can be as high as 27% [5].
409
2001 © A shley Publications Ltd. ISSN 1465-6566

410 Th e ph ar macology of meth otr ex ate

The treatmentof EPisstill primarily surgical, however, MTX is aviable and potentially superior alternative in selected patients. The medical management of EP allows the avoidance of surgery and its inherent morbidity. Success rates of medical management of EPwith MTXhave been demonstrated to approximate that of conservative surgical therapy, with no additional loss of future fertility. Recently, the American College of Obstetricians and Gynecologists has stated that medical treatment of EP with MTX is not an experimental therapy, but instead an effective, safe and cost-effective alternative [6].

2. Goal of therapy

EP is a life threatening condition that most clinicians will encounter in practice and, therefore, it is impera- tive to be familiarwith the latestmethodsof treatment. With the advent of quantitative hCG levels and trans- vaginal ultrasound monitoring, the detection of an abnormal gestation can be achieved much earlierand, therefore, patients tend to be diagnosed earlier in the course of their condition. Women who are clinically stable with a definitive diagnosis of EP less than 3.5 cmin diameter(by ultrasound), may be candidates for medical management with MTX [6].
The symptoms of abdominal pain and vaginal bleeding in the first trimester of pregnancy are the mostcommon clinical signs and symptoms suggestive of EP [7-10]. However, these symptoms are very common in early pregnancy. They may be presentin a threatened abortion or may be the result of other conditions such as cervical irritation, infection or trauma [7,9]. EP is a disease of the first trimester of pregnancy. Almost all ectopic pregnancies are diagnosed between 5and 12weeks of gestation. Due to the limited resolution of our current diagnostic techniques, the identification of EP much earlier than 5 weeks is rare [11,12]. By 12 weeks gestation, because of the enlargement of the uterus, an intra- uterine pregnancy should easily be distinguished froman EP. However, an EP of 12weeks gestation is a very serious condition that needs to be diagnosed promptly. What has become clearly apparent from research focused on EP is that an unruptured EP cannotbe confirmed solely on the presence of clinical symptoms and physical examination [7,8,10]. The diagnosis needs to be definitively made by following an algorithm using vaginal ultrasound, quantitative hCG and, in some cases, dilatation and curettage [7].

Extreme care must be taken not to treat a woman presumptively with MTX without ruling out the possibility of a completed miscarriage. Failure to visualise an intra-uterine pregnancy with ultrasound, when the patient has a quantitative hCG greater than an established discriminatory zone, does not confirm an EP. In fact, the diagnosis in this situation is more likely to be that of amiscarriage. Similarly, ultrasound identification of an adnexal masswithoutvisualisation of a definitive EP (gestational sac with a yolk sac or fetal cardiac activity) doesnotdefinitively diagnose an EP[13]. An increase (orplateau) in the serumhCG titer after dialation and evacuation (D&E) is considered definitive diagnosis of an EP [7]. Once a definitive diagnosis of EP has been made, the patient may be treated surgically or medically.
The goal of medical management with MTX is to selectively kill the rapidly dividing cells at the implan- tation site. The trophoblastis the active cell type atthe implantation site. Different sub populations of trophoblasts will both invade into the implantation site and secrete hCG. In the case of EP, these cells invade the fallopian tube epithelium and the underlying blood vessels resulting in rupture. If medical managementof an EPissuccessful, all growth of the ectopic gestation will cease and the body will spontaneously reabsorb any remaining products of conception and blood clot produced by the EP. The serum hCG concentration is used to monitor the success of treatment. Treatment is only considered successful when hCG is no longer detectable in the serum.

3. Available compounds
Currently, MTXis the only available agentused forthe medical management of EP. Each physician using MTX should understand this chemotherapeutic agent’s mechanism of action, its inherent risks and benefits and it’s side effect profile as it relates to dose and schedule of administration of the drug.

4. Mechanism of action
MTX is in aclass of drugs known as folic acid antago- nists [14]. This class of medications was the first to produce remission in leukaemia and the first cure of a solid tumour known as choriocarcinoma [15]. The method of action of MTX is to interrupt the synthesis of DNA. Folic acid is an essential component in the

synthesis of DNA precursors such as purines and thymidylate [14]. MTX and other folate analogues inactivate the enzyme dihydrofolate reductase (DHFR), which leads to the depletion of tetrahydrofo- late co-factors, required for DNA and RNA synthesis [14]. Crystallographic studies had revealed the atomic basis for high affinity of MTXfor DHFR and the differ- ences in amino acid sequences on the active centres of various DHFR that are responsible for the high specificity of the agents used in antimicrobial chemotherapy [14,17]. As folic acid analogues are polar, it has been shown that they only minimally cross the blood-brain barrier and do not get into the cerebrospinal fluid (CSF) in high enough concentra- tions to inhibit most cancer cells [14]. In order to trap folic acid analogues intracellularly, folyl polygluta- mate synthetase adds extra glutamate residues onto the molecule [14,17]. These residues do not cross cell membranes easily and, as such, this mechanism is efficient in increasing the intracellular concentration of drug, thus prolonging the action of the medication within the cells [14,17]. It is likely that this mechanism accounts for the feasibility of single-dose administra- tion of the drug.

Folate is reduced to tetrahydrofolate by DHFR by the addition of single carbon groups, which are then subsequently transferred in the synthesis of DNA and RNA [14]. Thymidylate synthase is the enzyme respon- sible for producing thymidylate, an essential component of DNA, from 2′-deoxyuridylate (dUMP) [14]. Tetrahydrofolate is converted to dihydrofolate when it donates a methyl group to dUMP in the production of thymidylate. In order to keep the reaction propagating, dihydrofolate must again be reduced by DHFR to tetrahydrofolate in order to continue donating methyl groups for subsequent reactions [14]. When DHFR is inhibited by folic acid analogues, the dihydrofolate polyglutamates build up in the cell and act as toxic substrates [14]. When this occurs, the one carbon transferreactionsare halted, as is the synthesis of DNA and RNA [14].

It has been known for many years that many of the chemotherapeutic agents act at specific stages of the cell cycle [14,18]. As MTXinterferes with the synthesis of DNA, cells in the S-phase of the cell cycle are most vulnerable to treatment. However, because MTX also interferes with RNA synthesis, it may also affect cells in the G1, G2, or G0 phases as synthesis of critical proteins for cell survival may also be impaired. Therefore, while the main action of MTX is on cells in

Bar n h ar t, Coutifar is & Esposito 411

the S-phase, many cells, regardlessof theirstage in the cell cycle, may suffer a lethal consequence.
As MTX is afolic acid antagonist, leucovorin rescue is a therapy that has revolutionised the use of MTX in tumours that usually require doses so high that prohibitive side effects would occur [16]. Leucovorin, also known as folinic acid, enters cells via the cell-specific carrier-mediated transport system [14,16]. Leucovorin does not require reduction by DHFR for conversion to other active folate co-factors [16]. In this way, it acts as arescue to cells whose DHFR has been inactivated by folic acid analogues.

5. Pharmacokinetics

MTX may be given orally, im., intrathecally or by continuous infusion. The two most common methods of administering MTX to patients with EP are the multiple-dose regimen of 1mg/kg of MTX alternating with 0.1 mg/kg of leucovorin rescue [10] and asingle- dose regimen based on body surface area (50mg/m ) without leucovorin rescue [19]. These two dosing regimens have been taken directly from large case series reported in the literature. The amount of MTX given per dose in these two regimens is similar. A woman of normal body surface area will receive slightly more MTX if dosed by body surface area than if dosed by mg/kg. Whatdiffers in these two regimens is the number and timing of the doses of MTX given, as well as the use of leucovorin (see Table 1 ).
There is a direct relationship between the dose and plasma concentrations and iv. administration of MTX. There is a triphasic disappearance of the drug [14]. The first is a rapid distributive phase, the second a renal clearance phase (half-life of 2 h) and a final phase in which the half-life is 8h. In this way, the action of MTX is prolonged. It is important to realise that when treating patients with renal failure the dose may have to be adjusted to reduce toxicity from excess build-up of the drug [14]. Around 35% of the drug is bound to plasma proteins and other drugs, such as sulfonamides, salicylates, tetracycline, chloramphenicol and phenytoin, may displace MTX fromthese plasma proteins and cause atoxic build-up of the drug [14,16]. The majority of MTX is excreted in the urine by the action of glomerial filtration and active tubular secretion [14]. Only a small fraction of MTXisexcreted through the biliary tract. Care mustbe taken in dosing MTX in patients with a reduction of

412 Th e ph ar macology of meth otr ex ate

Table 1: Administration and dosage of MTX to treat an ectopic pregnancy.
Single-dose Multi-dose

MTX
LEU

50 mg/m
None

2§

1 mg/kg
0.1 mg/kg

Frequency of dose One dose: Repeat in 1 week if necessary Up to 4 doses of each (MTX and LEU): Alternate
daily doses of MTX and LEU until serum hCG declines by 15%
Monitor hCG level Baseline (day 0), day 4 & day 7 Baseline (day 0), day 3, day 5 and day 7 until hCG
declines 15% from the previous value

When to administer additional

Second dose on day 7 if hCG value did not Give second, third, or fourth dose if hCG value has

doses

decline by 15% between days 4 and 7

not declined 15% from the prior value. Maximum 4 doses

Surveillance hCG (After initial
treatment response)

Weekly until hCG is not detectable Weekly until hCG is not detectable

Dose calculated by body surface area using nomogram.

hCG: human chorionic gonadotropin; LEU: Leucovorin; MTX: Methotrexate.

renal blood flow, in which cases excretion may be lessened, leading to toxicity.
MTX has many other therapeutic uses, alone or in combination with other medications. It has been shown to be effective in many conditions, including acute lymphoblastic leukaemia in children, treatment and prevention of adult leukaemia, choriocarcinoma and related trophoblast tumours, hydatidiform mole, osteosarcoma and mycosis fungoides, non-Hodgkin’s lymphoma and carcinomas of the breast, head and neck, ovary and bladder, psoriasis, prevention of graft versushost reactions from marrow transplant and the treatment of patients with rheumatoid arthritis.
As is seen with many chemotherapeutic preparations, cellular resistance may eventually develop and decrease the efficacy of the drug. Cancerous cells have many mechanisms by which they resist the action of antifolates, including the following [14]:
• impaired transport of MTX into cells
• production of altered forms of DHFR that have decreased affinity for the inhibitor

6. Current best practice

Medical management of EP is often used as primary therapy to avoid surgical intervention. MTX therapy can also be used to treat a persistent EP in the cases where all trophoblast cells have not been surgically removed fromthe tube afterlinearsalpingostomy [20]. The incidence of a persistent EP after conservative surgery ranges from 5- 10%. MTX can also be used prophylactically to preventpersistentEP atthe time of conservative surgical intervention [20,21]. There have been many uncontrolled studies reporting that systemic im. MTX therapy and laparoscopic salpin- gostomy have similar outcomes with respect to the rate of success of treatment, tubal patency and reproductive outcome [22-24]. It must be realised that while the short-term success of medical management of EP is comparable to surgical management, long-termpregnancy rates aftereach therapy have not been directly compared in arandomised clinical trail. While there are case reports describing a variety of MTX doses used for the treatment of EP, currently the only two commonly used regimens of MTX admini- stration are shown in Table 1 .

• increased concentrations of intracellular DHFR

6.1

Multi-dose administration

• decreased ability to synthesise MTX polyglutamates
• decreased thymidylate synthase activity.
Resistance to MTX is not a concern in the medical management of EP because of the short course of treatment.

The multi-dose regimen of MTX was the first to be described in large scale clinical practice [10]. In alarge case series published in 1991, MTX was given at a dose of 1 mg/kg im. In an attempt to lessen side effects, leucovorin was given in a dose of 0.1 mg/kg im. 24h later. A dose of MTXand adose of leucovorin, each given 24 h apart, is considered ‘one-dose’.

Subsequentdosesof MTXand leucovorin are continu- ously given until the hCG level has decreased by 15% on 2consecutive days [10]. A maximum of four doses can be given but all subjects do not require all four doses. Given the inter- and intra-variation of the hCG assay at the time, a 15% decline in serial hCG was considered definitive evidence of a decline in hCG production. Almost all algorithms have since adopted this cut-off. There has not been any investigation to test if a different cut off value in the decline in serial hCG could be used as a marker of successful treatment. The first large study published using this regimen noted success in 96 out of 100 patients treated. None of the 96 patients required more than four doses [10]. The four patients who failed therapy required surgery [10].

Bar n h ar t, Coutifar is & Esposito 413

studies using the single-dose regimen have noted the same success as the first study. A recent 1996 study noted a success rate of only 67% [25]. The side effects of therapy are similar to that of the multi-dose regimen.

6.3 Comparison of single- and multi-dose protocols
The efficacy of the single- and multi-dose protocols has never been directly compared in one trial. We have previously published acrude comparison of the data in the literature demonstrating that the success rate of these two protocols may not be equal. The success rate for the single-dose therapy is 88% with a range of 67 – 94% [26]. More than 15% of all patients

6.2

Single-dose administration

receiving the single-dose regimen actually received

A second commonly used regimen to treat EP is a single-dose regimen, which was introduced in 1993 [19]. This regimen utilises body surface area, rather than patient’s absolute weight, to calculate a dose. Dosing is calculated using the formula 50mg/m [19]. Leucovorin rescue is not given in this regimen. The follow-up of patients treated with this regimen is also different from the multi-dose regimen. In the case of the single-dose regimen, an hCG level is measured on the day of treatment and rechecked on days 4and 7 post-injection. If the hCG decline was less than 15% between days 4and 7then a repeat dose of MTX was given [19]. This cut-off was based on experience with the multi-dose regimen. Thus, the single-dose is a misnomer since the second weekly dose is often needed to achieve results similar to the multi-dose regimen. It is important to note that in many patients the hCG level may increase from the day of admini- stration (day 1) to day 4. It is possible that the single-dose of MTX has slowed the rise in hCG production, but not yet resulted in a decline. Therefore, this regimen relies on the body’s sponta- neous resolution of these damaged trophoblasts for full resolution to take place.
One advantage of this protocol is that the limited number of doses of MTXallows the elimination of the need for leucovorin rescue. Overall, there is also a reduction in the number of injections the patient receives compared to the multi-dose regimen. A total of 120 women with EP treated with a single-dose of MTX had a success rate of 94.2%. The seven patients who failed therapy all required surgical intervention [19]. It is important to note, however, that not all

more than one dose of MTX. The success for patients using the multi-dose therapy among the literature is 96% with a range of 89 – 100%. The difference between these rates in this meta-analysis was statisti- cally significant (p = < 0.05). It has been shown that the success rate of MTXis inversely related to the hCG level [27]. The higher the hCG level the greater possibility of failure of medical management with MTX. If the above comparison iscontrolled to account for differences in baseline hCG values among the different studies, the odds rate of failure with single- dose of MTX compared to a multi-dose regimen is 2.34with a95% confidence interval of 1.05- 5.23[26]. Stated differently, these data suggest that a woman treated with a single-dose therapy has a > 2-fold increase in her failure rate compared to women treated with the multi-dose protocol.

The percentage of women who experience side effects (nausea, vomiting, alopecia) is higher in women using multi-dose therapy (48%) than single- dose therapy (29%) [26]. The percentage of subjects who experience significantabdominal pain, however, was approximately equal (23% multi-dose and 20% single-dose) [26]. Currently, it is not known why patients experience abdominal pain after MTX injection. It is possible that this pain is a result of ischaemia of the fallopian tube, release of substances from the dying trophoblast cells or due to an enlarge- ment of the fallopian tube as a result of bleeding without tubal rupture. Patients with this pain should be followed very carefully to distinguish this known side effect from the possibility of tubal rupture.

414 Th e ph ar macology of meth otr ex ate

6.4 Indications and contraindications to MTX therapy
When given to carefully selected patients, medical management may avoid a surgical procedure with success rates similar to those of a linear salpingos- tomy. However, before one chooses to use single-dose or multi-dose therapy, a definitive diagnosis of EP must be made and it must be determined that a subject desires and is eligible for medical management. Because the medical manage- ment of EP is relatively new and there have been many published protocols, there is no absolute consensus on indication for therapy. However, medical management should be reserved for haemodynamically stable subjects who have been definitively diagnosed with a small, unruptured EP and who will be compliant with rigorous out-patient follow-up. MTX should not be administered to patients with a suspected ruptured EP or non-compliant patients who will not follow-up as directed [6,26]. The resolution of hCG to a negative value averages about 35 days, but may take up to 7 weeks [26].

Criteria for receiving MTX are listed in Table 2 . Relative contraindications to medical therapy include parameters that suggest that awoman is at high risk of treatment failure. These parameters include a high initial hCG level, the presence of fetal cardiac activity, or an adnexal mass (the entire mass, not just the gestational sac) of 3.5cm[6]. The treatment of women with these characteristics is not absolutely contraindi- cated, but the subject should understand that the success rate is expected to be lower. Importantly, only approximately 33- 40% of all patients diagnosed with an EPare eligible to receive medical management[26].

Contraindications to medical therapy are listed in Table 3 . MTXis contraindicated if there is evidence of immunocompromise, damage to organs that metabo- lise MTX or if the patient is afflicted with a condition that may be exacerbated by MTX [6]. Women should be screened with acomplete blood count (CBC), liver function tests and a serum creatinine. If a woman has a history of pulmonary disease, she should also be screened with a chest x-ray. There has been a case of fatal interstitial pneumonitis in subjects with underlying pulmonary disease after MTX administra- tion [26].

7. Side effects of MTX therapy
The side effects of MTX (Table 4 ) are limited by dose and duration of therapy [14]. The single-dose MTX regimen was started with the hope that it would reduce the adverse side effect profile. MTX tends to exert its powerful actions against rapidly dividing cells. The major areas of destruction are the rapidly dividing cells of the bone marrow and those of the intestinal tract. Destruction of bone marrow blood cell precursorspredisposespatientsto thrombocytopenia,
reticulocytopenia, lymphopenia and granulocy- topenia, which may result in life-threatening infections, severe anaemia and problems with sponta- neous haemorrhage [14]. These conditions have never been noted in the medical management of women with an EP with MTX. MTX may also cause haemor- rhagic enteritis, leading to nausea and vomiting, stomatitis, elevated liver function tests, anorexia, weight loss and bloody diarrhoea. These side effects have been noted in women treated for EP. MTX rarely causes nephrotoxicity, interstitial pneumonitis and alopecia dermatitis [14]. Fortunately, the experience

Bar n h ar t, Coutifar is & Esposito 415

with MTXtreatmentof EPhasyielded excellentresults with relatively few side effects. Two out of 100 patients suffered with stomatitis and three out of 100 patients had an elevated AST in the large study of the multi-dose regimen [10]. All symptoms resolved spontaneously [10]. Single-dose MTX has resulted in nausea and vomiting in one patient out of 120 [19].

Vaginal bleeding, abdominal pain and an initial rise in the serum hCG concentration are expected results of MTX treatment. The most important side effect of medical management is failure of treatment. Failed medical management is considered an emergency and surgical intervention is required (whether or not the tube has ruptured). Signs of treatment failure and tubal rupture include significantly worsening abdominal pain and haemodynamic instability regardless of change in hCG levels. Medical manage- ment is not considered successful until hCG is no longer detectable in the serum. If the serumhCG does not decline by at least 15% after one week from pre-treatment values, consideration should be given to the strong possibility of treatment failure. Increasing or plateauing β-hCG levels during the weeks following treatment is an indication to retreat with a second course of MTX or consider surgical management. Surgical intervention can also be used if the clinician suspects impending failure or if the patient no longer desires medical management and the stringent follow-up.

8. Conclusions
The medical management of EP with MTX is an importantadvancementin the treatmentof acommon and life threatening complication of the first trimester of pregnancy. Medical management of EP is cost-effective and reduces the morbidity associated with surgery while maintaining efficacy, safety and the potential forfuture fertility. The dose and duration of MTX therapy is much lower than that used for oncologic purposes, thus reducing side effects and increasing safety. Not all women with adiagnosed EP are candidates for medical management. As the efficacy of this therapy is not 100%, women must be followed until there is compete resolution of hCG titers from their serum.

9. Expert opinion
The medical management of EP with MTX is an example of a non-oncologic use of a chemothera- peutic agent. The use of MTX for this purpose is safe and effective. Simple protocols have been developed and can easily be followed. The ease of administra- tion, avoidance of surgical intervention and high success rates make the medical management of EP very attractive. However, most clinicians are not familiar with the administration of MTX. Often there are strict regulations regarding the facilities and expertise of the person administering this agent.

416 Th e ph ar macology of meth otr ex ate

These regulations may differ if the agent is given in a hospital or office setting. Additionally, there are pitfalls in the diagnosis and management of women with an EP. It is imperative to make the definitive diagnosis of an EP before the administration of MTX. Commonly the possibility of a miscarriage has not been definitively eliminated before initiation of treatment. In this case, a woman will have been exposed to a chemotherapeutic agent unnecessarily. MTX is considered an abortifacient and a potential teratogen. If an intra-uterine pregnancy is diagnosed after administration of MTX (regardless if it is viable), there are strong medical-legal implications.
Finally, there are no directcomparisons of the efficacy of the two commonly used protocols of MTX admini- stration. The single-dose is more commonly used because of its ease of administration and limited patientvisits. However, there is growing evidence that the multi-dose regimen has a higher success rate. It has been demonstrated that the success of medical management is inversely related to hCG level [27]. Extreme care should be taken when choosing a regimen of administration. Patient safety should not be sacrificed for convenience.

Bibliography
Papers of special note have been highlighted as:
• of interest
• • of considerable interest
1. CENTERS FOR DISEASE CONTROL AND PREVENTION: Ectopic Pr egn an cy – Un ited States, 1 990 – 1 992. MMWR (1995) 44 :46-48.
2. FYLSTRA D: Tubal pr egn an cy: a r eview of cur r en t diagn osis an d tr eatmen t. Obstet. Gynecol. Survey (1998) 53 :230-327.
3. NCHS: Advan ced r epor t of fin al mor tality statistics, 1 9 9 2 . Hy attsville, MD US Department of Health and Human Services, Public Health Services, CDC, 50 Monthly vital statistics report (1994) 43 (6) (Suppl).
4. CHOW WH, DALING JR, CATES W, GREENBERG RS: Epidemiology of ectopic pr egn an cy. Epidemiol. Rev. (1987) 9 :70-94.
5. MARCHBANKS PS, ANNEGERS J, COULAM C, STRATHY J, KURKLAND L: Risk factor s for ectopic pr egn an cy: A population -based study. JAMA (1988) 259 :1823-1827.
6. ACOG Practice Bulletin: Medical Man agemen t of Tubal Pr egn an cy. (Dec 1998):3 .
• The American College of Obstetricians and Gynecologists review of the medical management of ectopic pregnancy.
7. BARNHART K, MENNUTI M, BENJAMIN I, JACOBSON S, GOODMAN D, COUTIFARIS C: Pr om pt diagn osis of

ecto p ic p r egn an cy in an em er gen cy dep ar tm en t settin g. Obstet. Gynecol. (1994) 84 :1010-1015.
•• A concise description of a diagnostic algorithm used for the safe and timely diagnosis of women at risk for an ectopic pregnancy.
8. HIRATA AJ, SPIER DE, BUMP RC, HURT WG: Ectopic pr egn an cy in an ur ban teach in g h ospital: Can tubal r uptur e be pr edicted? SouthMed. J. (1991) 84 :1147-1149.
9. KADAR N, ROMERO R: Obser vation s on th e log h uman Ch or ion ic Gon adotr opin -time r elation sh ip in ear ly pr egn an cy an d its pr actical implication s. Am. J. Obstet. Gynecol. (1987) 1 57 :73-78.
10. STOVALLTG, LING FW, GRAY LA, CARSON SA, BUSTERJE: Me th o tr e x ate tr e atm e n t o f u n r u p tu r e d e cto p ic pr egn an cy: a r epor t of 1 00 cases. Obstet. Gynecol. (1991) 77 :749-753.
• Original case series describing the use of MTX in the treatment of ectopic pregnancy.
11. FLEISCHER AC, PENNELL RG, MCKEE MS et al.: Ectopic Pr egn an cy : featur es at tr an sv agin al so n o gr ap h y . Radiology (1990) 1 74 :375-378.
12. GOLDSTEIN SR, SNYDER JR, WATSON C, DANON M: Ver y e a r l y p r e gn a n c y d e t e c t i o n w i t h e n d o v a gi n a l ultr asoun d. Obstet. Gynecol. (1988) 72 :200-204.
13. BARNHART K, KAMELLE S, SIMHAN H: Diagn o stic accur acy of ultr asoun d, above an d below , th e βh CG d i s c r i m i n a t o r y z o n e . Obstet. Gynecol. (1999) 94 (4):583-587.
14. CALABRESI P, CHABNER BA: An tin eoplastic Agen ts, Ch ap 52. In: Goodman andGilman’sThePharmacological Basis of Therapeutics (Edition 8). Gilman A, Goodman LS, Goodman A (Eds), Macmillan Publishing Co., New York, NY, USA (1990):1275-1276.
• Complete review of the pharmacology of MTX and other chemotherapeutic agents.
15. FARBER S, DIAMOND LK, MERCER RD, SYLVESTER RF, WOLFF VA: Tempor ar y r emission s in acute leukemia in c h i l d r e n p r o d u c e d b y f o l i c a n t a go n i s t 4-ameth opter oylglutamic acid (amin opter in ). NEJM (1948) 238 :787-793.
16. HERTZ R: Folic acid an tagon ists: effects on th e cell an d th e patien t. Clin ical staff con fer en ce at N.I.H. Ann. Intern. Med. (1963) 59 :931-956.
17. CICHOWICZ DJ, SHANE B: Ma m m a l i a n f o l y l -y – polyglutamate syn th etase. 1 . Pur ification an d gen er al pr oper ties of th e h og liver en zyme. Biochemistry(1987) 26 :504-512.
18. SKIPPER HT, SCHABEL FM JR: Quan titative an d cytoki- n etic studies in ex per imen tal tumor models. In: Cancer Medicine (Edition 2). Holland JF, Frei E III (Eds), Lea & Febiger, Philadelphia, PA, USA (1982):663-684.
19. STOVALL TG, LING FW: Sin gle-dose meth otr ex ate: An ex pan ded clin ical tr ial. Am. J. Obstet. Gynecol. (1993) 1 68 :1759-1765.
• The case series first describing the use of the single-dose regimen for the treatment of ectopic pregnancy with MTX.

20. SEIFER DB: Per sisten t ectopic pr egn an cy: An ar gumen t for h eigh ten ed vigilan ce an d patien t com plian ce. Fertil. Steril. (1997) 68 (3):402-404.
21. SPANDORFER S, MENZIN A, BARNHART K, LIVOLSI V, PFEIFER S: Efficacy of fr ozen section evaluation of u te r i n e cu r e tti n gs i n th e d i a gn o s i s o f e cto p i c pr egn an cy. Am. J. Ob. Gyn. (1996) 1 75 :603-605.
22. FLORIDON C, THOMSEN SG: Meth otr ex ate tr eatmen t of ectopic pr egn an cy. Acta Obstet. Gynecol. Scand. (1994) 73 :746-752.
23. GOLDENBERG M, BIDER D, ADMON D, MASHIACH S, OELSNER G: Meth otr ex ate th er apy of tubal pr egn an cy. Hum. Reprod. (1993) 8 :660-666.
24. FYLSTRA D: Tubal pr egn an cy: a r eview of cur r en t diagn osis an d tr eatmen t. Obstet. Gynecol. Surv. (1998) 53 :230-327.
25. SITKA CS, ANDERSON L, FREDERIKSEN MD: Sin gle-dose meth otr ex ate for th e tr eatmen t of ectopic pr egn an cy:

Bar n h ar t, Coutifar is & Esposito 417
Nor th w ester n Memor ial Hospital th r ee-year ex per i-
en ce. Am. J. Obstet. Gynecol. (1996) 1 74 :1840-1846.
26. BARNHART KT, ESPOSITO M, COUTIFARIS C: An update on th e medical man agemen t of ectopic pr egn an cy. Obstet. Gynecol. Clin. North Am. (2000) 27 (3):653-667.
• A review of the medical management of ectopic pregnancy with a greater focus on the clinical aspects of diagnosis and treatment.
27. LIPSCOMB G, STOVALL T, LING W: No n s u r gical
t r e a t m e n t o f e c t o p i c p r e gn a n c y NEJM (2000) 343 :1325-1328.
• Mostrecentreview of the diagnosis and treatmentof ectopic pregnancy.

Kurt Barnhart , Christos Coutifaris & Melissa Esposito
Author for correspondence
Division of Human Reproduction, Department of Obstetrics and Gynecology, 106Dulles, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA, 19104, USA
Tel.: +1 215 662 2974; Fax: + 1 215 349 5512;
E-mail: [email protected]